MiRCURY LNA PCR System Manual

Expression Analysis
s, sample id u ic For bio se spec u e s a e pl l at manua a-pcr m/mirn o c . n o exiq
miRCURY LNA Universal RT microRNA PCR

Instruction manual v.5.2
#203301 March 2013
miRCURY LNA Universal RT microRNA PCR Instruction Manual
Table of contents
Product Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 I. Reagent kits. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 II. Primer Sets and Panels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Additional required materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Recommended accompanying products. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Control Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Before starting the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 A. Individual assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 B. Human and Mouse&Rat microRNA PCR Panels. . . . . . . . . . . . . . . . . . . . . . . . . . . 29 C. Focus microRNA PCR Panels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 D. Pick-&-Mix microRNA PCR Panels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 Tips to protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 Troubleshooting guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 FAQs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 Related products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Product summary
The miRCURY LNA Universal RT microRNA PCR system is a microRNA-specific, LNA-based system designed for sensitive and accurate detection of microRNA by quantitative real-time PCR using SYBR Green. The method is based on universal reverse transcription (RT) followed by real-time PCR amplication with LNA enhanced primers (for more details please see page 12). The miRCURY LNA Universal RT microRNA PCR portfolio is comprised of four types of reagent kits; including: Universal cDNA synthesis kit II RNA Spike-in kit ExiLENT SYBR Green master mix kit microRNA primer sets available in pre-dened Human, Mouse&Rat and Focus PCR panels and customized Pick-&-Mix PCR panels as well as individual primer sets and reference genes. All PCR panels are Ready-to-Use delivered with one 10 L PCR reaction per well.
Figure 1.
Universal cDNA synthesis kit II Description page 4
RNA Spike-in kit (optional)
+
microRNA and reference gene primer sets Pick-&-Mix PCR panels (Ready-to-Use) Predefined PCR panels Human, Mouse&Rat, and Focus PCR panels (Ready-to-Use)
Description page 6, protocol page 20
Description page 9, protocol page 43
Description page 7, protocol page 29+36
+
ExiLENT SYBR Green master mix 2,5 or 20ml
Description page 5
I. Reagent kits
Universal cDNA synthesis kit II, 8-64 rxns (product # 203301) This kit contains all reagents required for rst-strand cDNA synthesis for 8-64 reactions1). The UniSp6 RNA Spike-in template can be used by itself or in combination with the cel-miR-39-3p template provided in the RNA Spike-in kit:
Contents 5 x Reaction buffer2) Enzyme mix Nuclease free water UniSp6, RNA Spike-in template 128 L, 5 x concentrated 64 L, 10 x concentrated 1 ml 12 fmol, dried down
1)
Number of reactions is based on a standard reaction volume of 10 L to 80 L. Reaction volume depends on the application and number of assays to prole. Please consult Figure 4 for details. 2) Includes universal reverse transcription primer. 3) Used exclusively for the UniSp6 RNA Spike-in control primer set included in the ExiLENT SYBR Green master mix kit.
ExiLENT SYBR Green master mix, 2.5 ml (product# 203402) This kit contains all reagents required for PCR amplication of microRNAs. In addition, a positive control assay, the UniSp6 RNA Spike-in control primer set, is provided with this kit for amplication of the synthetic UniSp6 RNA spike-in provided in the Universal cDNA synthesis kit II. The reagents are provided in amounts sufcient for 500 reactions of 10 L.
Contents PCR Master mix Nuclease free water UniSp6 RNA Spike-in control primer set
1)
2 x 1.25 ml, 2x concentrated 2 x 1.25 ml 500 L, 10x concentrated
ExiLENT SYBR Green master mix, 20 ml (product# 203420) This kit contains all reagents required for PCR amplication of microRNAs. In addition, a positive control assay, the UniSp6 RNA Spike-in control primer set, is provided with this kit for amplication of the synthetic UniSp6 RNA spike-in provided in the Universal cDNA synthesis kit II (the UniSp6 RNA Spike-in control primer set is provided 100x concentration and should be diluted to 10x concentration before using in the protocol for individual assays). The reagents are provided in amounts sufcient for 4000 reactions of 10 L.
Contents PCR Master mix Nuclease free water UniSp6 RNA Spike-in control primer set
1)
2 x 10 ml, 2x concentrated 1 x 20 ml 500 L, 100x concentrated
1)
Used exclusively for detection of the UniSp6 RNA spike-in provided with the Universal cDNA synthesis kit II.
II. Primer Sets and Panels

MicroRNA LNA PCR primer set (product# 204000-205xxx) LNA PCR primer sets are designed for optimal performance with the Universal cDNA Synthesis Kit II and the ExiLENT SYBR Green master mix, kit II. The performance of LNA primer sets will be affected if used in combination with less than optimal reagents. The primer sets are supplied in sufcient amounts for 200 reactions of 10 L.
Contents LNA PCR Primer mix (dried down)
Reference gene primer set (product # varies) The Reference gene primer sets are designed for use with the microRNA primers sets above as reference genes for normalization. The primer mix is supplied in sufcient amount for 200 reactions of 10 L.
Contents LNA PCR Primer mix (dried down)
Pre-dened microRNA PCR Panels (product # varies) The Human, Mouse&Rat and Focus microRNA PCR panels Ready-to-Use consist of either 96-well or 384-well PCR plates containing dried down LNA primer sets for one 10 L realtime PCR reaction per well. The LNA primer sets are designed for optimal performance with the Universal cDNA Synthesis Kit II and the ExiLENT SYBR Green master mix, kit. The performance of the LNA primer sets will be affected if they are used in combination with less than optimal reagents. Contents Human miRNome panel I and II, V3 384-well PCR plates supplied with LNA primer sets dried down, one 10 L reaction per well:
Panel I 372 LNA primer sets for the amplication of human microRNAs1) 3 inter-plate calibrators 3 primer sets for reference genes
2) 3)
Panel II 380 LNA primer sets for the amplication of human microRNAs1) 3 inter-plate calibrators 1 blank well
5 RNA Spike-in control primer sets 1 blank well
Contents Mouse&Rat miRNome panel I and II, V3 384-well PCR plates supplied with LNA primer sets dried down, one 10 L reaction per well:
Panel I 372 LNA primer sets for the amplication of mouse and rat microRNAs1) 3 inter-plate calibrators 3 primer sets for reference genes
2) 3)
Panel II 380 LNA primer sets for the amplication of mouse and rat microRNAs1) 3 inter-plate calibrators 1 blank well
5 RNA Spike-in control primer sets 1 blank well
Contents Serum/Plasma Focus microRNA PCR panel, V3 PCR plates compatible with various real-time PCR instruments are available and supplied with LNA primer sets dried down, one 10 L reaction per well:
96-well (2 plates) 179 LNA primer sets for the amplication of human microRNAs1+2) 2x3 Inter-plate calibrators 5 RNA Spike-in control primer sets 2 blank wells - 1 in each plate
3)
384-well plate (2 panels per plate) 2x179 LNA primer sets for the amplication of human microRNAs1+2) 2x6 Inter-plate calibrators 2x5 RNA Spike-in control primer sets 3) 2x2 blank wells
Contents Cancer Focus microRNA PCR panel, V3 PCR plates compatible with various real-time PCR instruments are available and supplied with LNA primer sets dried down, one 10 L reaction per well:
96-well (1 plates) 84 LNA primer sets for the amplication of human microRNAs1) 3 Primer sets for potential reference genes2) 3 Inter-plate calibrators 5 RNA Spike-in control primer sets 1 blank well
3)
384-well plate (4 panels per plate) 4x84 LNA primer sets for the amplication of human microRNAs1) 4x3 Primer sets for potential reference genes2) 4x3 Inter-plate calibrators 4x5 RNA Spike-in control primer sets 3) 1 blank well
1)
Please go to www.exiqon.com/mirna-pcr to download plate layout les. Human Panels and Cancer Focus Panel: Three snRNAs (U6snRNA, SNORD38B, SNORD49A). Mouse&Rat Panels: Three snRNAs (U6snRNA, RNU5G, RNU1A1). Serum/plasma Focus Panel: miR-103, miR-191, miR-423-5p, miR-16, miR-425, miR-93, miR-451 are regarded reference gene candidates. 3) The RNA Spike-in control primer sets targets the UniSp6 RNA spike-in supplied in the Universal cDNA synthesis kit II and the 4 RNA spike-ins contained in the RNA Spike-in kit (UniSp2, UniSp4, UniSp5, and cel-miR-39-3p).
2)
Pick-&-Mix microRNA PCR Panel, (product # 203801 and 203802) The Pick-&-Mix microRNA PCR Panels consist of either 96-well PCR plates or 384-well PCR plates containing custom selections of dried down microRNA LNA PCR primer sets for one 10 L real-time PCR reaction per well, ready-to-use. PCR plates compatible with various real-time PCR instruments are available. The LNA primer sets are designed for optimal performance with the Universal cDNA Synthesis Kit II and the ExiLENT SYBR Green master mix kit. The performance of the LNA primer sets will be affected if they are used in combination with less than optimal reagents. Contents PCR plates supplied with customer dened LNA primer sets, reference gene primer sets, and RNA Spike-in control primer sets, dried down, one 10 L reaction per well1):
96-well PCR plates1) 10 primer sets in 8 replicates 22 primer sets in 4 replicates 92 primer sets in 1 replicate 96-well exible layout 384-well PCR plates1) 22 primer sets in 16 replicates 46 primer sets in 8 replicates 94 primer sets in 4 replicates 380 primer sets in 1 replicate 384-well exible layout
1)
Please go to www.exiqon.com/pick-and-mix to congure a Pick-&-Mix microRNA PCR Panel.
Storage
All microRNA PCR Panels, LNA primer sets and Reference gene primer sets The PCR panels and primer sets are shipped dried down at room temperature. The primers can be stored between +4C and -20C. Under these conditions, all components are stable for at least 12 months. After resuspension, it is recommended to store LNA primer sets and Reference gene primer sets in aliquots at -20C to avoid repeated freeze-thaw cycles. Universal cDNA synthesis kit II and ExiLENT SYBR Green master mix These kits are shipped on dry ice in polystyrene containers and should be stored at -15C to -25C. Do not store in a frost-free freezer. Under these conditions, all components are stable until the expiry date on the package or vial. It is recommended that the RNA spike-in be stored in aliquots at -20C after re-suspension to avoid repeated freeze-thaw cycles.
Additional required materials

Reagents not supplied ROX or other passive reference dye (required on some PCR cyclers) Materials and Equipment not supplied Nuclease-free PCR tubes or plates for use with individual assays Nuclease-free, aerosol barrier pipette tips Nuclease-free, low nucleic acid binding microcentrifuge tubes (e.g. Eppendorf DNA LoBind tubes product and original NUNC vials) Sealing foils for PCR plates Micro-centrifuge and plate centrifuge Heating block, thermal cycler or other incubators Real-time PCR instrument Optional but recommended product miRCURY LNA Universal RT microRNA PCR, RNA Spike-in kit (product# 203203)
10
Recommended accompanying products

Exiqon recommends the Exiqon GenEx qPCR software for comprehensive and convenient data analysis. GenEx includes a wizard for import of Exiqon miRCURY Universal RT microRNA PCR data and offers advanced methods to analyze real-time qPCR data in a few simple steps. The software includes tools for selection and validation of reference genes, data pre-processing and comprehensive statistical analyses. For more information and to download a free trial, please go to www.exiqon.com/qpcr-software. The following Exiqon GenEx products are available: Exiqon GenEx Industrial - Exiqon version of GenEx, qPCR analysis software, industrial license Exiqon GenEx Academic - Exiqon version of GenEx, qPCR analysis software, academic license Exiqon recommends the miRCURY RNA Isolation kits for purication of total RNA or small RNA fraction. RNA puried using the miRCURY RNA Isolation kits is fully compatible with the miRCURY LNA Universal RT microRNA PCR System. The following kits are available: miRCURY RNA Isolation Kit Cell & Plant Provides a rapid method for purication of total RNA from cultured animal cells, small tissue samples, blood, bacteria, yeast, fungi and plants. miRCURY RNA Isolation Kit Tissue Specically designed for purication of total RNA from tissue samples. miRCURY RNA Isolation Kit Biouids Kit for purication of low abundant small RNAs from samples such as serum, plasma, urine and CSF.
See Tip 2
11
Product description
A unique system for microRNA proling miRCURY LNA Universal RT microRNA PCR offers the best available combination of performance and ease-of-use on the microRNA real-time PCR market because it unites two important features (Figure 2): 1. Universal RT One rst-strand cDNA synthesis reaction provides template for all microRNA real-time PCR assays. This saves precious sample, reduces technical variation, requires use of less reagents, and saves time in the laboratory. 2. LNA PCR amplication Both PCR amplication primers (forward and reverse) are microRNA specic and optimized with LNA. The result is: 1) exceptional sensitivity as well as extremely low background enabling accurate quantication of very low microRNA levels and 2) highly specic assays that allow discrimination between closely related microRNA sequences. miRCURY LNA Universal RT microRNA PCR offers solutions both for high-throughput microRNA expression proling and for quantication of individual microRNAs.
Figure 2. Schematic outline of the miRCURY LNA Universal RT microRNA PCR System. A poly-A tail is added to the mature microRNA template (step 1A). cDNA is synthesized using a poly-T primer with a 3 degenerate anchor and a 5 universal tag (step 1B). The cDNA template is then amplied using microRNA-specic and LNA-enhanced forward and reverse primers (step 2A). SYBR Green is used for detection (step 2B).
Step 1: First-strand synthesis (RT)

Mature microRNA A) B) AAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAA TTTTTTTTTTTTTTT 3 degenerate anchor 5 universal tag
Step 2: Real-time PCR amplification

miR-specific forward primer A) TTTTTTTTTTTTTTT miR-specific reverse primer
B)
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Control Assays
There are 3 different types of control assays available in the miRCURY LNA Universal RT microRNA PCR system: Reference assays and reference candidates Inter-plate calibrators RNA spike-in assays All of these control assays are available in the microRNA PCR panels. One RNA spike-in template is provided with the Universal cDNA Synthesis Kit II, while the assay that will detect this RNA spike-in is available in the ExiLENT SYBR Green master mix. Additionally 4 RNA spike-in templates are available as a spike-in kit. The assays for detection of these 4 templates as well as the reference assays are available as individual primer sets. Reference assays and reference candidates These assays detect small non-coding RNAs - either small nuclear RNA, small nucleolar RNA or microRNA which frequently are found to be stably expressed across different cells or tissues. Reference assays may therefore be candidate assays for normalization in a proling study with several samples. Though this is a good and recommended approach, great caution should be taken in the selection of reference genes. The danger of using endogenous reference genes lies in the assumption that a specic gene is expressed at the exact same level in all sample types. This is rarely true. The selection of reference genes should therefore be made with care, and should be specic to the sample set you are working with. The actual selection of reference genes to be used for normalization should always be based on a determination of the most stably expressed gene(s) which may be done using either GeNorm or NormFinder both tools that are integrated within Exiqons GenEx data analysis software. When applicable, we recommend using microRNA rather than small nuclear RNA or small nucleolar RNA for normalization. Firstly, small nuclear and nucleolar RNAs are longer RNA species than microRNA and may purify differently from microRNA. Moreover, small nuclear and nucleolar RNAs have entirely different functions as well as sub-cellular locations, and nally certain samples like blood plasma does not contain the small nuclear and nucleolar RNAs. Global mean normalization is a preferred alternative to using reference genes for normalization when working with panels and samples where many microRNAs are screened per sample and where many microRNA are called (detected) in all samples. Exiqons GenEx will easily perform global mean normalization. To read more on normalization.
See Tip 11
13
Inter-plate calibrators Three wells within the pre-dened Human, Mouse&Rat, and Focus PCR Panels contain the inter-plate calibrator assay (annotated as UniSp3 IPC in the plate layout les). Depending on the plate layout, the Pick-&-Mix Panels contain at least three inter-plate calibrators. Each of these wells, contain a pre-aliquoted primer pair and a DNA template and therefore the variation is very minimal from well-to-well and from plate-to-plate of these assays. The inter-plate calibrators are used for calibration between PCR plate runs which is very useful on some instruments that apply the cycle threshold method for Cq determination such as the ABI7900 PCR cycler. Since the inter-plate calibrators are independent of cDNA quality in order to give a signal (but may be affected by PCR inhibitors in the sample) they may be used to quality control each plate run. Inter-plate calibration (IPC) can easily be performed in the data analysis software Exiqons GenEx. Alternatively, IPC may be performed manually by using the IPC assay replicates as follows. For each plate, verify that the replicates have Cq standard deviation within 0.5. If this is not the case, eliminate the outlier if this can be identied. Calculate the average of the replicates for each plate, the overall average (average of IPC values from all plates). The calibration factor is calculated as the difference between plate average and overall average for each plate (calibration factor = IPCplate-IPCoverall). An example is shown in Table 1. Finally, calibrate each plate by subtracting the calibration factor from all Cq values in the plate.
Table 1. Example of inter-plate calibration (IPC)
Plate 1 let-7c IPC plate average IPC overall average Calibration factor let-7c calibrated 21.12 19.72 19.81 -0.09 21.21
Plate 2 20.93 19.70 19.81 -0.11 21.04
Plate 3 21.34 20.00 19.81 0.19 21.15
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RNA spike-ins (synthetic control templates) The primary purpose of the RNA spike-ins and the matching primer pairs for detection of these is to provide controls for the quality of the RNA isolation, the cDNA synthesis reaction and the PCR. RNA isolations may vary in yield, purity and integrity. Some sample types may contain compounds that inhibit the cDNA synthesis or the PCR even though the RNA has been puried using the best standard procedures. This may result in different efciencies of the reverse transcription or PCR between compared samples. One way to control for differences in efciencies at each experimental level (isolation, cDNA synthesis, and PCR) is by adding known RNA spike-ins to the sample prior to isolation and cDNA synthesis. Use of the RNA spike-ins may also reveal if nucleases are present. After conducting the PCR but before initiating the data analysis, wells detecting RNA spike-ins are compared and outlier samples may be identied and considered for exclusion in the further data analysis. We have designed a ight of RNA spike-ins for this purpose. The UniSp6 RNA Spike-in template is provided with the Universal cDNA synthesis kit II. Additionally four RNA spikein templates can be obtained with the separate RNA Spike-in kit. Here, a vial of three RNA spike-in templates, UniSp2, UniSp4 and UniSp5, mixed at different concentrations can be used during RNA isolation. The cel-miR-39-3p RNA template provided in a separate vial in the RNA Spike-in kit can be mixed with the UniSp6 template from the Universal cDNA synthesis kit II to obtain two different template concentrations. This combination can be added during the cDNA synthesis. Five wells in pre-dened PCR panel plates contain the matching primer sets. The selection of RNA Spike-in control primer set in the Pick-&-Mix PCR Panel can be customized to the specic need. A UniSp6 control primer set is also provided with the ExiLENT SYBR Green master mix kit, which is to be used with our non-plate based PCR primer set products.The RNA spike-ins are shipped dried down and must be re-suspended before use. If the RNA Spike-in kit with multiple RNA spike-ins is used, follow the protocol accompanying that product. If UniSp6 is to be used alone: 1. Re-suspend the UniSp6 RNA spike-in by adding 80 L nuclease free water to the tube. 2. Mix by vortexing and spin down. Leave for 20-30 min. on ice to fully dissolve RNA spike-in. Mix by vortexing and spin down. Store in aliquots at -20C 3. Prior to the RT reaction, add 1 L synthetic spike-in (10 8 copies/L) per 20 ng sample RNA.
15
An alternative application of the UniSp6 RNA spike-in is as inter-plate calibrator. This is only relevant when using individual LNA primer sets and Reference gene primer sets in a multi-plate set-up. The microRNA PCR panels already contain an inter-plate calibrator. Add 1 L synthetic spike-in (10 8 copies/L) to 20 ng of a complex RNA sample (e.g. total RNA from MS2, yeast, or a cell line; not provided with the kit). Proceed with rst strand synthesis and subsequently real-time PCR as described in the protocols of the current instruction manual. At least one spike-in amplication reaction per PCR plate is used for inter-plate calibration.
See Tip 9
Experimental design Before starting the experiment, it is essential to consider the experimental setup and consider the number of replicates needed for obtaining signicant results replicates being technical as well as biological. The number of biological replicates required varies from experiment to experiment depending on the variation within and between the groups. We recommend that a No Template Control (NTC) is included in the study every time a new experiment is set up, to set the background level. The most optimal NTC is a mock up sample preparation including only carrier RNA as sample. The NTC should be run on all assays included in the study. We dene the background of an assay as 5 Cq values below the NTC level. Furthermore we recommend including spike-ins found in the Spike-in kit to provide full quality control over all steps in the proling (see gure 3). Finally it is necessary to include a number of candidate reference miRNAs, which are expected to be constitutively expressed across the different experimental conditions, for data normalization.
Figure 3. Experimental design
Sold as separate Spike-in kit (203203)
Comes with Universal cDNA synthesis kit II cel-miR-39-3p UniSp6
Included in all panel products
Assays available as individual assays or in ready-to-use panels miR-A miR-X
UniSp2 Sample n Sample n+1 NTC
UniSp4
UniSp5
UniSp3
Sample preparation evaluation
cDNA synthesis evaluation
Inter plate Calibration
Sample profiling
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Before starting the Experiment

Important note RNA work requires specic handling and precautions to prevent RNase contamination of the reagents and degradation of the RNA sample. Find information on how to handle RNA in the tips section starting on page 52. The tips section also provides simple guidelines for good laboratory practice to ensure optimal performance of PCR experiments.
Before setting up a real-time PCR experiment, there are a number of practical experimental design parameters that should be considered: RNA input - The miRCURY LNA Universal RT microRNA PCR protocol is optimized for use of 20 ng total RNA per 20L cDNA synthesis reaction. The exact amount of total RNA needed depends on whether the downstream application is individual assays or panels. Furthermore, the amount of total RNA to be used may also vary depending on the microRNA expression levels in the cells or tissue to be analyzed. For highly expressed microRNAs it is possible to use down to 10 pg total RNA as starting material. For weakly expressed microRNAs it may be possible to use up to 200 ng of total RNA; however, in samples with high amounts of PCR inhibitors (e.g. FFPE tissue samples), this may not be feasible. Finally, inhibitors may be present in RNA preparations from certain samples e.g. serum and plasma. Prior to conducting a larger microRNA proling study, it is recommended to optimize the amount of input RNA to the RT reaction in order to avoid conducting a larger study where inhibition occurs sporadically throughout the data set. Information on how to extract and handle RNA can be found in the tips section. In short, total RNA should be prepared using a method that preserves small RNA species. DNase treatment may be necessary. When using commercially available kits, please ensure that the total RNA preparation is guaranteed to contain microRNAs.
17
Note: Blood serum and plasma are particular sample types that require special RNA purication procedures and the amount of RNA present in the samples can usually not be accurately determined. Due to the low levels of microRNAs and potentially high levels of inhibitors in samples derived from serum and plasma, specic recommendations for how to set up experiments using these types of sample can be found in the miRCURY LNA Universal RT microRNA PCR, Instruction Manual Biouid samples. Normalization when running individual assays or when conguring a Pick-&-Mix microRNA PCR panel it is important to consider how the data will be normalized. For tips and recommendations on choosing the correct reference genes and how to test and validate reference genes, please see section on reference assays (page13) and the Tip 11 (page 58).
See Tip 11
Excess volumes required for pipetting Liquid handling with pipettes or pipetting robots require excess volumes of reagents due to loss during pipetting. The loss depends on the available pipetting system but losses in the range from 10% -25% are not uncommon. All protocols in the current instruction manual reect the required reaction volumes and pipetting volumes should be adjusted according to accommodate the pipetting loss of the available pipetting system. ROX ROX is a passive reference dye used by some PCR cycles to obtain a robust read over the entire array of wells in a 96- or 384-well PCR plate. The requirement for ROX is instrument dependent and we recommend to follow the instrument manufactures guidelines on this (see also Tip 8).
See Tip 8
ABI instruments the default settings on ABI real-time PCR cyclers are not suitable for running miRCURY LNA Universal RT microRNA PCR. Settings need to be changed from automatic to manual background and threshold settings to obtain valid PCR data (see also Tip 10). Furthermore, if the dataset is to be analyzed using the GenEx software, it is important that the experiment is set up as an AQ experiment, not RQ. To ensure correct settings, download the instrument settings le at www.exiqon.com/sds.
See Tip 10
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RNA spike-ins consider how the RNA spike-ins should be applied in the planned study. Please consult the section on RNA spike-ins on page 15. Protocols for the rst-strand cDNA synthesis and real-time amplication follows on the next pages: A. Individual assays, please go to page 20 B. Human and Mouse&Rat microRNA PCR Panels, please go to page 29 C. Focus microRNA PCR Panels, please go to page 36 D. Pick-&-Mix microRNA PCR Panels, please go to page 43 Figure 4 gives an overview and helps identifying which protocol to follow as well as the recommended cDNA reaction volume needed for a given sample and assay type.
Figure 4. Protocol overview
Sample type Panels or primer sets: Universal cDNA reactions per kit Universal cDNA reaction volume Dilution of cDNA in ExiLENT Master Mix
Cells or tissue miRNome panel I miRNome panel I+II Pick-&-Mix/Focus 1-96 97-192 Individual assays (<96)
Serum plasma or other biofluids miRNome panel I miRNome panel I+II Pick-&-Mix/Focus 1-96 97-192 Individual assays (<96)
32
16
64
32
64
16
64
32
64
20 L
40 L
10 L
20 L
10 L
40 L
80 L
10 L
20 L
10 L
100x
80x
50x
40x
Use standard printed manual or download from www.exiqon.com/pcr-manual
Use biofluids manual from www.exiqon.com/pcr-manual-biofluids
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Protocol A - Individual assays

This protocol is used for conducting the rst-strand cDNA synthesis and real-time PCR, using the individual assays for human, mouse and rat (product numbers 204000-205xxx). If working with serum plasma samples or other biouids, please refer to the specic miRCURY LNA Universal RT microRNA PCR Instruction Manual for biofluid samples at www.exiqon.com/serum-plasma-pcr-manual. Before using the LNA PCR primer mix or the Reference gene primer mix for the rst time, the primers must be re-suspended: Re-suspend the primer mix by adding 220 L nuclease free water to the tube. Mix by vortexing and spin down. Leave on ice for 20-30 minutes. The UniSp6 RNA spike-in control primer mix is supplied with the master mix and is already dissolved. Ensure primer concentration is 10x before proceeding with the protocol (see page 5 for details). Additional required materials: 96- or 384-well plate real-time PCR cycler Thermocycler for rst-strand cDNA synthesis 96/384-well plates or tube strips compatible with available real-time PCR cycler Micro centrifuge Swing bucket centrifuge for 96-/384-well plates
Protocol Individual assays 20
Checklist: Have you considered excess volumes required for using liquid handling robotics see page 18 Did you consider how to use the RNA spike-ins please see page 15 ROX: The ExiLENT SYBR Green master mix, does not include the ROX passive reference dye. Please follow instrument manufactures recommendations ABI instruments: The use of manual background and threshold settings is necessary for obtaining correct PCR data. Make sure to have the optimal settings by downloading the instrument settings le at www.exiqon.com/sds. Furthermore, if the data is to be analyzed using GenEx, the experiment must be set up as an AQ experiment, not RQ Have you optimized the input amount to the RT reaction in order to avoid inhibition?
Workow for individual primer sets (per sample)
Phase I: Prepare RNA sample See page 52 for recommendations
Phase II: cDNA synthesis See protocol page 23.
Phase III: real-time PCR amplification See protocol page 25. - Prepare adequate amount of pre-mixed primer + PCR Master mix and distribute into wells - Add cDNA to all primer sets to be analyzed
Protocol Individual assays
LNATM primer sets
Relative expression (log2)
4 3 2 1 0 -1
Normal Tumor Total Tumor
miR-21 let-7a
Tumor stroma
Phase IV: Data analysis See data analysis guide online - Export data for further analysis - Data pre-processing, normalization and statistical analysis
22
Protocol The miRCURY LNA Universal RT microRNA PCR protocol is a two-part protocol consisting of: 1. First-strand cDNA synthesis (Step 1-5) 2. Real-time PCR amplication (Step 6-11) Important: Keep reagents and reactions on ice (or at 4C) at all times. First strand synthesis
Step 1 Dilute template RNA Adjust each of the template RNA samples to a concentration of 5 ng/L using nuclease free water.
Step 2 Prepare reagents
Gently thaw the 5x Reaction buffer and nuclease-free water, and immediately place on ice. Mix by vortexing. Re-suspend the RNA spikeins according to the appropriate RNA Spike-ins protocol (see page 15), leave on ice for 15-20 minutes. Immediately before use, remove the Enzyme mix from the freezer, mix by icking the tubes and place on ice. Spin down all reagents.
Step 3 Combine reagents according to Table 2 Note: remember to calculate necessary excess volume for pipetting and robotic dead volume.
If performing rst-strand cDNA synthesis on multiple RNA samples, it is recommended to prepare an RT working solution of the 5x Reaction buffer, water, Enzyme mix and RNA spike ins (in the proportion indicated in the rst four lines of Table 2). The following procedure is recommended: 1. Prepare the required amount of RT working solution and place it on ice. 2. Dispense RT working solution into nuclease free tubes. 3. Dispense template RNA in each tube.
Table 2 Reverse transcription reaction setup Reagent 5x Reaction buffer Nuclease-free water Enzyme mix Synthetic RNA spike ins, optional replace with H2O if omitted Template total RNA (5 ng/L) Total volume Volume (L), RT reaction 2 4.5 1 0.5 2 10
Step 4 Mix and spin reagents
Mix the reaction by very gentle vortexing or pipetting to ensure that all reagents are thoroughly mixed. After mixing, spin down.
Step 5 Incubate and heat inactivate1)
Incubate for 60 min at 42C. Heat-inactivate the reverse transcriptase for 5 min at 95C. Immediately cool to 4C. Store at 4C or freeze.
1)
The protocol can be interrupted at this stage. The undiluted cDNA may be kept at -20C for up to 5 weeks (optional store at 4C for up to 4 days). It is recommended that synthesized cDNA is stored in low-nucleic acid binding tubes or plates.
24
qPCR protocol
Step 6 Prepare reagents for real-time PCR Place cDNA (from Step 5), nuclease free water and PCR Master mix on ice and thaw for 15-20 min. Protect the PCR Master mix vials from light. Immediately before use, mix the PCR Master mix by pipetting up and down. The rest of the reagents are mixed by vortexing and spun down.
Step 7 Dilute cDNA template 80x in nuclease free water2)
Immediately before use, dilute only the amount of cDNA template needed for the planned real-time PCR reactions 80x in nuclease free water (e.g. add 395 L nuclease free water to each 5 L of reaction). It is important that low-nucleic acid binding tubes or plates are used. It is not recommended to store the 1:80 dilution of cDNA. Recommendation: Include a passive reference dye in the cDNA dilution if advised by instrument manufacturer. Please note that the PCR Master mix does not include ROX. The amount of ROX required is instrument dependent and it is important to refer to the manufacturers recommendations when deciding how much ROX to use, see Tip 8.
See Tip 8
2)
Adjust volumes to accommodate your in-house liquid handling system volume loss when pipetting
25
Step 8 Combine PCR Master mix, PCR primer mix and cDNA according to Table 3. Note: remember to calculate necessary excess volume for pipetting and robotic dead volume.
When multiple real-time PCR reactions are performed with the same microRNA primer set, it is recommended to prepare a primer master mix working-solution of the PCR primers and the PCR Master mix (in the proportion indicated in Table 3). The following procedure is recommended: 1. Prepare the required amount of primer:master mix working-solution (see Table 3) and place it on ice. It is recommended to include excess of all reagents in the master mix to compensate for pipetting excess material. 2. Place the relevant volume of primer:master mix working-solution in PCR tubes/wells (see Table 3) and spin tubes/plate briey in a centrifuge (1500g for 1 minute), to remove air bubbles. 3. Add cDNA template to each tube/well.
Table 3 Real-time PCR reaction, pr. 10 L reaction3) Reagent PCR Master mix PCR primer mix4) Diluted cDNA template Total volume Volume (L), 96/384-well plate, tubes or strips 5 1 4 10
Mix the reaction by gentle pipetting to ensure that all reagents are mixed thoroughly. After mixing cap tubes or strips or seal the plate with optical sealing as recommended by the manufacturer. Spin down in a centrifuge (1500g for 1 minute). The experiment can be paused at this point. Store the reactions protected from light at 4C for up to 24 hours.
3)
If using a 96-well cycler with a minimum recommended volume of 20 L (as some ABI instruments), then use 10 L reaction volume and set the instrument settings at 20 L. 4) The PCR primer mix must be dissolved prior to real-time PCR set-up, see page 20.
26
ABI instrument user?

Apply manual baseline and threshold settings! Go to www.exiqon.com/sds to download settings le
Step 10 Real-time PCR amplication
Perform real-time PCR amplication followed by melting curve analysis according to Table 4.
Table 4 Real-time PCR cycle conditions Process step Polymerase Activation/Denaturation Amplication Settings, LC480 instrument5) 95C, 10 min 45 amplication cycles at 95C, 10 s 60C, 1 min, ramp-rate 1.6C/s 6) Optical read Yes Settings, other instruments 3) 95C, 10 min 40 amplication cycles at 95C, 10 s 60C, 1 min, ramp-rate 1.6C/s 6) Optical read Yes
Melting curve analysis7)
Step 11 Analyze data
Perform initial data analysis using the software supplied with the realtime PCR instrument to obtain raw Cq values (Cp or Ct, depending on PCR instrument). If you are using an ABI instrument, please note that it is not recommended to use auto Ct settings. Furthermore, if the data is to be analyzed using Exiqon GenEx, the experiment must be set up as an AQ experiment, not RQ. For a guide on how to set manual baseline and threshold, refer to Tip 10, page 56 in the tips section. If you are using a Roche LC480 instrument, we recommend analysis using the 2nd derivative method. For tips on nor malization, please see T ip 11, page 5 8. We recommend performing normalization and further data analysis with the Exiqon GenEx qPCR analysis software (w w w.exiqon. com/mirna-pcr-analysis). Please refer to our data analysis guide for recommendations.
See Tip 11
5)
Five additional ampli cation cycles are required when using the LC480 instrument to allow collection of assay data with Cp-values up to 40. 6) The ramp-rate of cooling from 95C to 60C should be set to 1.6C/s. This is equivalent to 100% under standard cycling conditions on the ABI 7500, 7900 and Viia7 instruments. If the ramp rate of cooling is too rapid, performance may be compromised. 7) Melting curve analysis of the PCR product(s) is recommended to verify specicity and identity of the amplication reaction. Melting curve analysis is an analysis step built into the software of instruments. Please follow the instructions provided by the supplier. Note: The Tm of a PCR product depends on buffer composition, salt concentration and the PCR instrument.
Protocol B - Human and Mouse&Rat microRNA PCR Panels

This protocol is used for conducting the rst-strand cDNA synthesis and real-time PCR using the following products: Human miRNome PCR Panel (product numbers 203611 to 203614) Mouse&Rat miRNome PCR Panel (product numbers 203709 to 203712) If working with serum plasma samples or other biouids, please refer to the speci c miRCURY LNA Universal RT microRNA PCR Instruction Manual for biouid samples at www.exiqon.com/serum-plasma-pcr-manual. Additional required materials: 384-well plate real-time PCR cycler Thermocycler for rst-strand cDNA synthesis Micro centrifuge Tube for mixing water and master mix (10 ml) Sealing foils for PCR plates Swing bucket centrifuge for 96/384-well plates Recommended: Liquid handling robot for pipetting Checklist: Have you considered excess volumes required for using liquid handling robotics see page 18 Did you consider how to use the RNA spike-ins please see page 15 ROX: The ExiLENT SYBR Green master mix, does not include the ROX passive reference dye. Please follow instrument manufactures recommendations ABI instruments: The use of manual background and threshold settings is necessary for obtaining correct PCR data. Make sure to have the optimal settings by downloading the instrument settings le at www.exiqon.com/sds. Furthermore, if the data is to be analyzed using GenEx, the experiment must be set up as an AQ experiment, not RQ. Have you optimized the input amount to the RT reaction in order to avoid inhibition?
Protocol - Human, Mouse&Rat Panels 29
Workow for Human and Mouse&Rat microRNA PCR Panels (per sample)
Phase III: real-time PCR amplification See protocol page 33. - Mix cDNAs with PCR Master mix - Add cDNA:PCR Master mix to PCR plates
Protocol - Human, Mouse&Rat Panels
4 3 2 1 0 -1
miR-21 let-7a
Tumor stroma
30
Protocol The miRCURY LNA Universal RT microRNA PCR protocol is a two-part protocol consisting of: 1. First-strand cDNA synthesis (Step 1-5) 2. Real-time PCR amplication (Step 6-9) Important: Keep reagents and reactions on ice (or at 4C) at all times. First strand synthesis:
Step 1 Dilute template RNA Adjust each of the template RNA samples to a concentration of 5 ng/l using nuclease free water.
Gently thaw the 5x Reaction buffer and nuclease-free water, and immediately place on ice. Mix by vortexing. Re-suspend the RNA spikein(s) according to the appropriate RNA Spike-in protocol (see page 15), leave on ice for 15-20 minutes. Immediately before use, remove the Enzyme mix from the freezer, mix by icking the tubes and place on ice. Spin down all reagents.

31
If performing rst-strand cDNA synthesis on multiple RNA samples, it is recommended to prepare an RT working solution of the 5x Reaction buffer, water, Enzyme mix and RNA spike ins (in the proportions indicated in the rst four lines of Table 5). The following procedure is recommended: 1. Prepare the required amount of RT working solution and place it on ice. 2. Dispense RT working solution into nuclease free tubes. 3. Dispense template RNA in each tube.
Table 5 Reverse transcription reaction setup Reagent 5x Reaction buffer Nuclease-free water Enzyme mix Synthetic RNA spike ins, optional replace with H2O if omitted Template total RNA (5ng/L) Total volume Panel I Volume (L) 4 9 2 1 4 20 Panel I+II Volume (L) 8 18 4 2 8 40
1)
32
qPCR protocol:
Step 7 Combine PCR Master mix, water and cDNA and add to PCR plates2)
The following procedure is recommended to avoid low concentrations of cDNA from adhering to tube surface: 1. Before removing the plate seal, briey spin down the plate(s) in a plate centrifuge. 2. Combine 2x PCR Master mix and water. Panel I: 2000 L 2x master mix and 1980 L water, Panel I+II: 4000 L 2x master mix and 3960 L water. 3. Mix gently and spin down. 4. Add 20 L cDNA (panel I) or 40 L cDNA (panel I+II) and mix. 5. Add 10 L PCR Master mix: cDNA mix to each well 3). 6. Seal the plate with optical sealing as recommended by the instrument manufacturer. 7. Spin plate briey in a plate centrifuge (1500g for 1 minute), to to collect the sample. The experiment can be paused at this point. Store the reactions protected from light at 4C for up to 24 hours. Recommendation: Include a passive reference dye in the cDNA dilution if advised by instrument manufacturer. Please note that the PCR Master mix does not include ROX. The amount of ROX required is instrument dependent and it is important to refer to the manufacturers recommendations when deciding how much ROX to use, see Tip 8.
See Tip 8

Table 6 Real-time PCR cycle conditions
Process step Polymerase Activation/Denaturation Amplication
Settings, LC480 instrument 4) 95C, 10 min 45 amplication cycles at 95C, 10 s 60C, 1 min, ramp-rate 1.6C/s 5) Optical read Yes
Settings, other instruments 95C, 10 min 40 amplication cycles at 95C, 10 s 60C, 1 min, ramp-rate 1.6C/s 5) Optical read Yes
Melting curve analysis 6)
Step 9 Analyze data
Perform initial data analysis using the software supplied with the realtime PCR instrument to obtain raw Cq values (Cp or Ct, depending on PCR instrument). If you are using an ABI instrument, please note that it is not recommended to use auto Ct settings. For a guide on how to set manual baseline and threshold, refer to Tip 10, page 56 in the tips section. Furthermore, if the data is to be analyzed using Exiqon GenEx, the experiment must be set up as an AQ experiment, not RQ. Alternatively, use ABI settings les available from www.exiqon.com/sds. If you are using a Roche LC480 instrument, we recommend analysis using the 2nd derivative method. For tips on normalization, please see Tip 11, page 58. We recommend performing normalization and further data analysis with the Exiqon GenEx qPCR analysis software (www.exiqon.com/mirna-pcr-analysis). Please refer to our data analysis guide for recommendations.
See Tip 11
34
2) 3)
Adjust volumes to accommodate your in-house liquid handling system and the inaccuracy these have when pipetting. Corresponding to 0.05ng total RNA starting material pr. PCR reaction. 4) Five additional amplication cycles are required when using the LC480 instrument to allow collection of assay data with Cp-values up to 40. 5) The ramp-rate of cooling from 95C to 60C should be set to 1.6C/s. This is equivalent to 100% under standard cycling conditions on the ABI 7500, 7900 and Viia7 instruments. If the ramp rate of cooling is too rapid, performance may be compromised. 6) Melting curve analysis of the PCR product(s) is recommended to verify specicity and identity of the amplication reaction. Melting curve analysis is an analysis step built into the software of instruments. Please follow the instructions provided by the supplier. Note: The Tm of a PCR product depends on buffer composition, salt concentration and the PCR instrument.
Protocol C - Focus microRNA PCR Panels

This protocol is used for conducting the rst-strand cDNA synthesis and real-time PCR, using the Cancer Focus microRNA PCR panels, 96-well plates (product numbers 203832 to 203835) and 384-well plates (product numbers 203840 and 203841). If working with serum plasma samples or other biouids, please refer to the specic miRCURY LNA Universal RT microRNA PCR Instruction Manual for biouid samples at www.exiqon.com/serum-plasma-pcr-manual. Additional required materials: 96- or 384-well plate real-time PCR cycler Thermocycler for rst-strand cDNA synthesis Micro centrifuge Tube for mixing water and master mix (10 ml) Sealing foils for PCR plates Swing bucket centrifuge for 96/384-well plates Recommended: Liquid handling robot for pipetting Checklist: Have you considered excess volumes required for using liquid handling robotics see page 18 Did you consider how to use the RNA spike-ins please see page 15 ROX: The ExiLENT SYBR Green master mix does not include the ROX passive reference dye. Please follow instrument manufactures recommendations ABI instruments: The use of manual background and threshold settings is necessary for obtaining correct PCR data. Make sure to have the optimal settings by downloading the instrument settings le at www.exiqon.com/sds. Furthermore, if the data is to be analyzed
Protocol Focus Panels
using GenEx, the experiment must be set up as an AQ experiment, not RQ Have you optimized the input amount to the RT reaction in order to avoid inhibition?
36
Workow for Focus microRNA PCR Panels (per sample)
Phase III: real-time PCR amplification See protocol page 40. - Mix cDNAs with PCR Master mix - Add cDNA:PCR Master mix to PCR plates
4 3 2 1 0 -1
miR-21 let-7a
Tumor stroma
37
Protocol Focus Panels 38
When performing rst-strand cDNA synthesis on multiple RNA samples, it is recommended to prepare an RT working solution of the 5x Reaction buffer, water, Enzyme mix and RNA spike ins (in the proportions indicated in the rst four lines of Table 7). The following procedure is recommended: 1. Prepare the required amount of RT working solution and place it on ice. 2. Dispense RT working solution into nuclease free tubes. 3. Dispense template RNA in each tube. Table 7 Reverse transcription reaction setup
Reagent 5x Reaction buffer Nuclease-free water Enzyme mix Synthetic RNA spike ins, optional replace with H2O if omitted Template total RNA (5ng/L) Total volume Per panel Volume (L) 2 4.5 1 0.5 2 10
1)
39
qPCR protocol:
Step 7 Combine PCR Master mix, water and cDNA and add to PCR plates2)
The following procedure is recommended to avoid low concentrations of cDNA from adhering to tube surface: 1. Before removing the plate seal, briey spin down the plate(s) in a plate centrifuge. 2. Combine 2x PCR Master mix and water: 1000 L 2x master mix and 990 L water (Focus panel consisting of 2x 96 assays) 3. Mix gently and spin down. 4. Add 10 L cDNA (Focus panel consisting of 2x 96 assays). 5. Add 10 L PCR Master mix: cDNA mix to each well 3). 6. Seal the plate with optical sealing as recommended by the instrument manufacturer. The experiment can be paused at this point. Store the reactions protected from light at 4C for up to 24 hours. Recommendation: Include a passive reference dye in the cDNA dilution if advised by instrument manufacturer. Please note that the PCR Master mix does not include ROX. The amount of ROX required is instrument dependent and it is important to refer to the manufacturers recommendations when deciding how much ROX to use, see Tip 8.
See Tip 8
2) 3)
Adjust volumes to accommodate your in-house liquid handling system and the losses these have when pipetting. Corresponding to 0.05ng total RNA starting material pr. PCR reaction.
40

Step 8 Spin plate
Spin plate briey in a plate centrifuge (1500g for 1 minute), to collect the sample.
Table 8 Real-time PCR cycle conditions

Process step Polymerase Activation/Denaturation Amplication Settings, LC480 instrument5) 95C, 10 min Settings, other instruments 4) 95C, 10 min
45 amplication cycles at 95C, 10 s 60C, 1 min, ramp-rate 1.6C/s 6) Optical read Yes
40 amplication cycles at 95C, 10 s 60C, 1 min, ramp-rate 1.6C/s 6) Optical read Yes
Melting curve analysis7)
4)
If using a 96-well cycler with a minimum recommended volume of 20 L (like some ABI instruments), then use 10 L reaction volume and set the instrument settings at 20 L. 5) Five additional amplication cycles is required when using the LC480 instrument to allow collection of assay data with Cp-values up to 40. 6) The ramp-rate of cooling from 95C to 60C should be set to 1.6C/s. This is equivalent to 100% under standard cycling conditions on the ABI 7500, 7900 and Viia7 instruments. If the ramp rate of cooling is too rapid, performance may be compromised. 7) Melting curve analysis of the PCR product(s) is recommended to verify specicity and identity of the amplication reaction. Melting curve analysis is an analysis step built into the software of instruments. Please follow the instructions provided by the supplier. Note: The Tm of a PCR product depends on buffer composition, salt concentration and the PCR instrument.
41
Perform initial data analysis using the software supplied with the realtime PCR instrument to obtain raw Cq values (Cp or Ct, depending on PCR instrument). If you are using an ABI instrument, please note that it is not recommended to use auto Ct settings. Furthermore, if the data is to be analyzed using Exiqon GenEx, the experiment must be set up as an AQ experiment, not RQ. For a guide on how to set manual baseline and threshold, refer to Tip 10, page 56 in the tips section. If you are using a Roche LC480 instrument, we recommend analysis using the 2nd derivative method. For tips on normalization, please see Tip 11, page 58. We recommend performing normalization and further data analysis with the Exiqon GenEx qPCR analysis software (www.exiqon.com/mirna-pcr-analysis). Please refer to our data analysis guide for recommendations.
See Tip 11
Protocol Focus Panels 42
Protocol D - Pick-&-Mix microRNA PCR Panels

This protocol is used for conducting the rst-strand cDNA synthesis and real-time PCR, using the Pick-&-Mix microRNA PCR Panel, 96-well plates (product number 203801) and 384-well plates (product number 203802). If working with serum plasma samples or biouid samples, please refer to the specic miRCURY LNA Universal RT microRNA PCR Instruction Manual for biouid samples at www.exiqon.com/serum-plasma-pcr-manual. Additional required materials: 96- or 384-well plate real-time PCR cycler Thermocycler for rst-strand cDNA synthesis Micro centrifuge Tube for mixing water and master mix (10 ml) Swing bucket centrifuge for 96/384-well plates. Sealing foils for PCR plates Recommended: Liquid handling robot for pipetting Checklist: Have you considered excess volumes required for using liquid handling robotics see page 18 Did you consider how to use the RNA spike-ins please see page 15 ROX: The ExiLENT SYBR Green master mix does not include the ROX passive reference dye. Please follow instrument manufactures recommendations ABI instruments: The use of manual background and threshold settings is necessary for obtaining correct PCR data. Furthermore, if the data is to be analyzed using GenEx, the experiment must be set up as an AQ experiment, not RQ. Make sure to have the optimal settings by downloading the instrument settings le at www.exiqon.com/sds. Have you optimized the input amount to the RT reaction in order to avoid inhibition?
Protocol Pick-&-Mix Panels 43
Workow for Pick-&-Mix PCR plates (per sample)
Phase III: real-time PCR amplification See protocol page 48. - Mix cDNAs with PCR Master mix - Add cDNA:PCR Master mix to primer sets replicates (indicated by colored boxes)
Protocol Pick-&-Mix Panels
4 3 2 1 0 -1
miR-21 let-7a
Tumor stroma
44
Pick-&-Mix microRNA PCR Panel layouts Overview of the pre-dened plate layouts available for 96-well and 384-well plates of the Pick-&-Mix Panel. Wells in dark gray and light greay are pre-occupied by interplatecalibrators (UniSp3 IPC) and RNA spike-in controls (UniSp6), respectively.
22 microRNAs x 16 samples
1
A B C D E F G H I J K L M N O P
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
2
2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
3
3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
4
4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4
5
5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5
6
6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6
7
7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7
8
8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 17 17 17 17 17 17 17 17 17 17 17 17 17 17 17 17 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 22 22 22 22 22 22 22 22 22 22 22 22 22 22 22 22 CP CP CP CP CP CP CP CP CP CP CP CP CP CP CP CP IPC IPC IPC IPC IPC IPC IPC IPC IPC IPC IPC IPC IPC IPC IPC IPC
1
A B C D E F G H
1 1 1 1 1 1 1 1
1 2
2 26 2 26 2 26 2 26 2 26 2 26 2 26 2 26
3
3 27 3 27 3 27 3 27 3 27 3 27 3 27 3 27
4
4 28 4 28 4 28 4 28 4 28 4 28 4 28 4 28
5
5 29 5 29 5 29 5 29 5 29 5 29 5 29 5 29
6
6 30 6 30 6 30 6 30 6 30 6 30 6 30 6 30
7
7 31 7 31 7 31 7 31 7 31 7 31 7 31 7 31
8
8 32 8 32 8 32 8 32 8 32 8 32 8 32 8 32
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
9 33 9 33 9 33 9 33 9 33 9 33 9 33 9 33 10 34 10 34 10 34 10 34 10 34 10 34 10 34 10 34 11 35 11 35 11 35 11 35 11 35 11 35 11 35 11 35 12 36 12 36 12 36 12 36 12 36 12 36 12 36 12 36 13 37 13 37 13 37 13 37 13 37 13 37 13 37 13 37 14 38 14 38 14 38 14 38 14 38 14 38 14 38 14 38 15 39 15 39 15 39 15 39 15 39 15 39 15 39 15 39 16 40 16 40 16 40 16 40 16 40 16 40 16 40 16 40 17 41 17 41 17 41 17 41 17 41 17 41 17 41 17 41 18 42 18 42 18 42 18 42 18 42 18 42 18 42 18 42 19 43 19 43 19 43 19 43 19 43 19 43 19 43 19 43 20 44 20 44 20 44 20 44 20 44 20 44 20 44 20 44 21 45 21 45 21 45 21 45 21 45 21 45 21 45 21 45 22 46 22 46 22 46 22 46 22 46 22 46 22 46 22 46 23 47 23 47 23 47 23 47 23 47 23 47 23 47 23 47 CP IPC CP IPC CP IPC CP IPC CP IPC CP IPC CP IPC CP IPC
2
2 2 2 2 2 2 2 2
3
3 3 3 3 3 3 3 3
4
4 4 4 4 4 4 4 4
5
5 5 5 5 5 5 5 5
6
6 6 6 6 6 6 6 6
7
7 7 7 7 7 7 7 7
8
8 8 8 8 8 8 8 8
9 10 11 12
9 9 9 9 9 9 9 9 10 10 10 10 10 10 10 10 CP CP CP CP CP CP CP CP IPC IPC IPC IPC IPC IPC IPC IPC
1 25 1 25 1 25 1 25 1 25 1 25 1 25 1 25
1
A B C D E F G H
1 13 1 13 1 13 1 13
1 2
1 1 13 13 25 25 37 37 49 49 61 61 73 73 85 85
3
2 2 14 14 26 26 38 38 50 50 62 62 74 74 86 86
4
2 2 14 14 26 26 38 38 50 50 62 62 74 74 86 86
5
3 3 15 15 27 27 39 39 51 51 63 63 75 75 87 87
6
3 3 15 15 27 27 39 39 51 51 63 63 75 75 87 87
7
4 4 16 16 28 28 40 40 52 52 64 64 76 76 88 88
8
4 4 16 16 28 28 40 40 52 52 64 64 76 76 88 88
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
5 5 17 17 29 29 41 41 53 53 65 65 77 77 89 89 5 5 17 17 29 29 41 41 53 53 65 65 77 77 89 89 6 6 18 18 30 30 42 42 54 54 66 66 78 78 90 90 6 6 18 18 30 30 42 42 54 54 66 67 78 78 90 90 7 7 19 19 31 31 43 43 55 55 67 67 79 79 91 91 7 7 19 19 31 31 43 43 55 55 67 67 79 79 91 91 8 8 20 20 32 32 44 44 56 56 68 68 80 80 92 92 8 8 20 20 32 32 44 44 56 56 68 68 80 80 92 92 9 9 21 21 33 33 45 45 57 57 69 69 81 81 93 93 9 9 21 21 33 33 45 45 57 57 69 69 81 81 93 93 10 10 22 22 34 34 46 46 58 58 70 70 82 82 94 94 10 10 22 22 34 34 46 46 58 58 70 70 82 82 94 94 11 11 23 23 CP CP 47 47 59 59 IPC IPC 83 83 95 95 11 11 23 23 CP CP 47 47 59 59 IPC IPC 83 83 95 95 12 12 24 24 36 36 48 48 60 60 72 72 84 84 96 96 12 12 24 24 36 36 48 48 60 60 72 72 84 84
2
2 14 2 14 2 14 2 14
3
3 15 3 15 3 15 3 15
4
4 16 4 16 4 16 4 16
5
5 17 5 17 5 17 5 17
6
6 18 6 18 6 18 6 18
7
7 19 7 19 7 19 7 19
8
8 20 8 20 8 20 8 20
9 10 11 12
9 21 9 21 9 21 9 21 10 22 10 22 10 22 10 22 11 23 11 23 11 23 11 23 IPC CP IPC CP IPC CP IPC CP
1 1 13 13 25 25 37 37 49 49 61 61 73 73 85 85
96 96
92 microRNAs x 1 sample
1
A B C D E F G H
1 13 25 37 49 61 73 85
380 microRNAs x 1 sample

1 2
2 26 50 74 98 122 146 170 194 218 242 266 290 314 338 362
3
3 27 51 75 99 123 147 171 195 219 243 267 291 315 339 363
4
4 28 52 76 100 124 148 172 196 220 244 268 292 316 340 364
5
5 29 53 77 101 125 149 173 197 221 245 269 293 317 341 365
6
6 30 54 78 102 126 150 174 198 222 246 270 294 318 342 366
7
7 31 55 79 103 127 151 175 199 223 247 271 295 319 343 367
8
8 32 56 80 104 128 152 176 200 224 248 272 296 320 344 368
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
9 33 57 81 105 129 153 177 201 225 249 273 297 321 345 369 10 34 58 82 106 130 154 178 202 226 250 274 298 322 346 370 11 35 59 83 107 131 155 179 203 227 251 275 299 323 347 371 12 36 60 84 108 132 156 180 204 228 252 276 300 324 348 372 13 37 61 85 109 133 157 181 205 229 253 277 301 325 349 373 14 38 62 86 110 134 158 182 206 230 254 278 302 326 350 374 15 39 63 87 111 135 159 183 207 231 255 279 303 327 351 375 16 40 64 88 112 136 160 184 208 232 256 280 304 328 352 376 17 41 65 89 113 137 161 185 209 233 257 281 305 329 353 377 18 42 66 90 114 138 162 186 210 234 258 282 306 330 354 378 19 43 67 91 115 139 163 187 211 235 259 283 307 331 355 379 20 44 68 92 116 140 164 188 212 236 260 284 308 332 356 380 21 45 69 93 117 141 165 189 213 237 261 285 309 333 357 381 22 46 70 94 118 142 166 190 214 238 262 286 310 334 358 382 23 47 71 95 119 143 167 191 215 239 263 287 311 335 359 383 24 48 72 IPC 120 144 168 IPC 216 240 264 CP 312 336 360 IPC
2
2 14 26 38 50 62 74 86
3
3 15 27 39 51 63 75 87
4
4 16 28 40 52 64 76 88
5
5 17 29 41 53 65 77 89
6
6 18 30 42 54 66 78 90
7
7 19 31 43 55 67 79 91
8
8 20 32 44 56 68 80 92
9 10 11 12
9 21 33 45 57 69 81 93 10 22 34 46 58 70 82 94 11 23 35 47 59 71 83 95 IPC CP IPC IPC 60 72 84 96
1 25 49 73 97 121 145 169 193 217 241 265 289 313 337 361
45
Protocol Pick-&-Mix Panels 46
Step 3 Combine reagents according to Table 9
When performing rst-strand cDNA synthesis on multiple RNA samples, it is recommended to prepare an RT working solution of the 5x Reaction buffer, water, Enzyme mix and RNA spike ins (in the proportions indicated in the rst four lines of Table 9). The following procedure is recommended: 1. Prepare the required amount of RT working solution and place it on ice. 2. Dispense RT working solution into nuclease free tubes. 3. Dispense template RNA in each tube. Table 9 Reverse transcription reaction setup per sample1)
Reagent
Volume (L) < 100 miRNA analyzed per sample 2 4.5 1 0.5 2 10
Volume (L) > 100 miRNA analyzed per sample 4 9 2 1 4 20
5x Reaction buffer Nuclease-free water Enzyme mix Synthetic RNA spike ins, optional replace with H2O if omitted Template total RNA (5ng/L) Total volume
The amount of 100x fold diluted cDNA needed for the different Pick-&-Mix layouts can be seen in Table 10 Step 7.
1)
The amount of cDNA required per sample depends on the number of microRNAs analyzed per sample. The volumes suggested here provides excess amount of cDNA, which ensures the highest possible reproducibility because these volumes can be pipetted with great accuracy.
47
qPCR protocol:
2)
Although not recommended, the protocol can be interrupted at this stage. The undiluted cDNA may be kept at -20C for up to 5 weeks (optional store at 4C for up to 4 days). It is recommended that synthesized cDNA be stored in low-nucleic acid binding tubes or plates.
48
Step 7 Dilute cDNA template 100x in nuclease free water3)
Dilute the cDNA from the RT reactions to give a nal 100x dilution. Suggested cDNA dilution procedures is shown in Table 10 along with required volumes of diluted cDNA for the different Pick-&-Mix predened layouts. It is receommeded that low-nucleic acid binding. For fully customized layouts dilutions may be adjusted to the specic replicate scheme. tubes or plates are used. It is not recommended to store the 1:100 dilution of cDNA. Table 10 Amount of 100x diluted cDNA needed in Pick-&-Mix plates
Pick-&-Mix plate conguration
Suggested cDNA dilution procedure cDNA + nuclease free water (L) 2 + 198 2 + 198 5 + 495 2 + 198 3 + 297 5 + 495 20 + 1980
Volume (L) of diluted cDNA needed for each Pick-&-Mix plate
8x10 (12) 4x22 (24) 1x92 (96) 16x22 (24) 8x46 (48) 4x94 (96) 1x380 (384)
60 120 480 120 240 480 1920
Numbers in parenthesis designate total number of assays/sample, including controls and inter-plate calibrators. Loss from pipetting is not included in the volumes. listed in the right column (diluted cDNA needed). Recommendation: Include a passive reference dye in the cDNA dilution if advised by instrument manufacturer. Please note that the PCR Master mix does not include ROX. The amount of ROX required is instrument dependent and it is important to refer to the manufacturers recommendations when deciding how much ROX to use, see Tip 8.
See Tip 8
3)
Adjust volumes to accommodate your in-house liquid handling system and the losses these have when pipetting.
49

Step 8 Combine cDNA and PCR Master mix 1:1 and add to PCR plates
The following procedure is recommended: 1. Before removing the plate seal, briey spin down the plate(s) in a plate centrifuge. 2. Combine 2x PCR Master mix and 100x diluted cDNA 1:1 (e.g. 500 L 2x PCR master mix and 500 L diluted cDNA). 3. Mix gently by inverting the tube, spin down. 4. Add 10 L PCR Master mix: cDNA mix to each well.4) 5. Seal the plate with optical sealing as recommended by the instrument manufacturer. The experiment can be paused at this point. Store the reactions protected from light at 4C for up to 24 hours.
Step 9 Spin plate
Spin plate briey in a plate centrifuge (1500g for 1 minute), to remove air bubbles.
Perform real-time PCR amplication followed by melting curve analysis according to Table 11. Table 11 Real-time PCR cycle conditions
Process step Polymerase Activation/Denaturation Amplication
Settings, LC480 instrument5) 95C, 10 min 45 amplication cycles at 95C, 10 s 60C, 1 min, ramp-rate 1.6C/s7) Optical read Yes
Settings, other instruments 6) 95C, 10 min 40 amplication cycles at 95C, 10 s 60C, 1 min, ramp-rate 1.6C/s7) Optical read Yes
Melting curve analysis 8)
50
Perform initial data analysis using the software supplied with the realtime PCR instrument to obtain raw Cq values (Cp or Ct, depending on PCR instrument). If you are using an ABI instrument, please note that it is not recommended to use auto Ct settings. For a guide on how to set manual baseline and threshold, refer to Tip 10, page 56 in the tips section. Furthermore, if the data is to be analyzed using Exiqon GenEx, the experiment must be set up as an AQ experiment, not RQ. For tips on normalization, please see Tip 11, page 58. If you are using a Roche LC480 instrument, we recommend analysis using the 2nd derivative method. We recommend performing normalization and further data analysis with the Exiqon GenEx qPCR analysis software (www.exiqon.com/mirna-pcr-analysis). Please refer to our data analysis guide for recommendations.
See Tip 11
4) 5)
Corresponding to 0.05ng total RNA starting material pr. PCR reaction. Five additional amplication cycles are required when using the LC480 instrument to allow collection of assay data with Cp-values up to 40. 6) If using a 96-well cycler with a minimum recommended volume of 20 L (like some ABI instruments), then use 10 L reaction volume and set the instrument settings at 20 L. 7) The ramp-rate of cooling from 95C to 60C should be set to 1.6C/s. This is equivalent to 100% under standard cycling conditions on the ABI 7500, 7900 and Viia7 instruments. If the ramp rate of cooling is too rapid, performance may be compromised. 8) Melting curve analysis of the PCR product(s) is recommended to verify specicity and identity of the amplication reaction. Melting curve analysis is an analysis step built into the software of instruments. Please follow the instructions provided by the supplier. Note: The Tm of a PCR product depends on buffer composition, salt concentration and the PCR instrument.
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Tips to protocol
RNA extraction and template preparation Tip 1. RNA extraction and template preparation Purication and preparation of total RNA that includes small RNAs (<200 nt) from a biological sample is the rst critical step for a successful expression proling analysis of microRNAs. Therefore, the method used for RNA sample preparation is critical to the success of the experiment. The following points should be considered before starting the experiment: We recommend using the miRCURY RNA Isolation kit for isolation of RNA that contains the small RNA fraction. If not using one of the miRCURY RNA Isolation kits, it is important that the method used to isolate RNA from a sample should give a quantitative recovery of small RNAs and should not result in a substantial loss of the small RNA fraction. This also applies when using commercially available kits. Make sure the total RNA preparation is guaranteed to contain microRNA. If commercially available puried RNA is used, it is important to make sure that the RNA is guaranteed to contain small RNAs and is representative of the microRNAs in the species and/or tissue from which it was isolated. The comparison of samples prepared using different RNA isolation methods is not recommended. However, if this is necessary, it is recommended to include the measure of a reference small RNA which has a consistent and unvaried expression level in order to allow for comparison of microRNA levels in the different sample preparations. The isolation of RNA and the reaction steps preceding real-time PCR should be performed in rooms separate from where real-time PCR experiments will take place in order to avoid contamination with amplicon. It is recommended that the integrity of isolated RNA be assessed before proceeding with quantitative real-time PCR experiments. This may be performed either on the Agilent 2100 Bioanalyzer or by denaturing gel electrophoresis. If necessary, treat RNA preparations with DNases to remove contaminating DNA that may interfere with the real-time PCR results. Puried total RNA should be dissolved in nuclease-free water at a stock concentration of at least 1 g/L. See recommendations for storage conditions in Tip 3.
52
Tip 2. Guidelines for serum, plasma and biouid samples Plasma and serum are essentially cell free liquid samples. This means that only circulating RNA is extracted from these sample types, resulting in low total RNA concentrations, even if the microRNA fraction is readily detectable. The result of this is that measuring correct RNA concentrations is difcult, and that there is a high risk of increased loss during extraction. For this reason, we recommend using RNA amounts based on starting volume rather than RNA quantity. Comprehensive guidelines for microRNA proling in blood serum/plasma can be downloaded at www.exiqon.com/serum-plasma-guidelines.
Tip 3. General guidelines for handling and storage of RNA The following precautions should be taken to prevent RNase contamination and degradation of the RNA sample and reagents: Always wear disposable gloves. Use nuclease-free, low nucleic acid binding plasticware and lter barrier pipette tips. Keep tubes capped when possible. Always spin tubes before opening. For long-time storage, RNA may be stored at -80C. Avoid repeated freezing and thawing cycles.
Tip 4. Good PCR laboratory practice To reduce the risk of contaminating PCRs with old PCR amplicons, and consequently obtain false results: Always wear a clean lab coat. Use separate lab coats for RNA sample preparation, cDNA synthesis and when setting up PCR reactions or handling PCR products. Change gloves often, especially whenever you suspect they may have been contaminated. Establish and maintain designated areas for PCR set-up, PCR amplication, and gel electrophoresis of PCR products. Never bring amplied PCR products into the PCR set-up area. Spin down all reaction and sample tubes before opening. Open and close all reagent and sample tubes carefully, trying not to splash or spray PCR samples. Keep reactions and components capped whenever possible. Use lter barrier pipette tips to avoid aerosol-mediated contamination of your pipetting device. Clean laboratory benches and equipment regularly.
53
Tip 5. Keep reagents and reactions cool at all times In order to ensure optimal performance of the miRCURY LNA Universal RT microRNA PCR system it is important that the reagents and reactions are kept on ice (or at 4C) as much as possible during the protocol (apart from reaction steps specically involving raised temperatures).
Tip 6. First strand cDNA synthesis Store cDNA samples in nuclease-free low nucleic acid-binding micro centrifuge tubes, e.g. Eppendorf DNA LoBind tubes.
Tip 7. Recommended controls The following controls are recommended and should be included in the experimental set-up: Reverse transcription/no enzyme controls, i.e. rst-strand cDNA synthesis reactions performed without the Enzyme mix. If a PCR product is amplied from this control reaction it indicates genomic DNA or PCR product contamination of the template RNA. Non-template controls in the real-time PCR amplication, i.e. real-time PCR reactions performed without any cDNA template. This control will reveal PCR product contamination of the reaction. Blank purication or carrier only, i.e. when purifying RNA in the presence of carrier RNA (such as for serum and plasma samples). This control will reveal any non-specic signals originating from the carrier RNA alone.
Tip 8. Passive reference dye (ROX) Many real-time PCR instruments will only produce reliable results when a passive reference dye such as ROX is added to the PCR reaction. The reference dye is used to normalize signals from individual PCR wells in order to enable comparison of real-time PCR amplication signals across an entire PCR-plate. It is recommended to determine whether your real-time PCR instrument has this type of requirement. The amount of ROX to include in the PCR reaction depends on the requirements of the real-time PCR instrument and must be adjusted accordingly. Please follow the suppliers instructions for preparation and concentrations of ROX solutions. Typically, real-time PCR instruments that allow excitation at individual wavelengths for individual dyes (most lter wheel based instruments) require less ROX than instruments that use a single excitation wavelength for all uorophores (most laser based instruments use excitation at 488 nm).
54
It is important to note that excessive amounts of ROX may inhibit the PCR reaction. It may be recommended to perform a titration of ROX amounts in order to determine the optimal concentration for a particular instrument-system combination. It is possible to use the spike-in template (provided in the Universal cDNA synthesis kit II) and the Control primers (provided in the ExiLENT SYBR Green master mix kit) to perform such titrations.
Tip 9. Inter-plate calibration When setting up large scale experiments with several PCR plates involved, individual run differences may be observed. One way of avoiding these having an effect on the results is setting up the experiment in such a way that all samples for one gene are run on the same plate. However, this is not always feasible. When Cq values for one gene have to be compared across plates, it is recommended to employ an inter-plate calibrator. An inter-plate calibrator is a template-assay combination which is the same in all plates, and always located in the same well across different plates. This can then be used to calibrate all plates to give the same Cq value for the calibrator, thereby reducing run-to-run variance. Please note that all panels contain inter-plate calibrators.
55
Tip 10. Guidelines for real-time PCR data collection using ABI instruments On cyclers using baseline and threshold values for Cq (Ct) calculations, such as ABI 7900HT, it is important that the proper settings are used. Use of the automatic function of the software for these settings does not seem to produce optimal results for SYBR Green based assays. Often the baseline is set erroneously on non-detected assays, and this in turn gives false positives, therefore do not use automatic settings. Another issue to consider when using automatic settings is that the settings may differ between plates resulting in data that cannot be compared directly. Inter-plate calibration may not fully resolve this issue, since each assay has a separately calculated baseline and threshold. Instead, both threshold and baseline should be set manually, applying the same settings for all assays on the plate. The following principles should be applied to manual baseline and threshold settings: Baseline: The baseline should be calculated in the cycle interval before the amplication takes off (see Figure 3). Threshold: The threshold should then be set with the Y-axis in log scale where all assays are in the log linear phase, and the threshold above background for all assays (see Figure 3). Note: The optimal threshold value may vary between individual machines and experiments.
Important note If ROX passive reference dye has not been used in the PCR reactions, make sure the SDS software is set-up without reference dye correction.
56
Figure 5.
Threshold above background
Amplication take-off
Baseline interval
57
Tip 11. Quick guide to normalization of microRNA qPCR experiments The purpose of normalization is to remove technical and biological variation between samples that is not related to the biological changes under investigation. Proper normalization is critical for the correct analysis and interpretation of results from real-time PCR experiments. The most commonly used option for normalization is to use stably expressed reference genes. In general it is recommended to test several endogenous control candidates (reference genes) before setting up the actual microRNA expression analysis. These candidates should be chosen among genes that can be expected to be stably expressed over the whole range of samples being investigated. They can be stably expressed small non-coding RNA, or stably expressed microRNAs, and chosen based on literature or pre-existing data (e.g. microarray analysis or qPCR panel screening). Exiqon offers primer sets for a number of different small RNAs which tend to be stably expressed, and are therefore often good candidates for reference genes. Its important to keep in mind that in spite of being small non-coding RNAs, most of these are signicantly larger than microRNA and therefore may have different extraction efciency and stability. U6 is one such reference gene which is often used. However, U6 is signicantly larger than microRNAs and has a different sub-cellular distribution. The existence of several different isoforms also makes it a suboptimal reference gene. 5S ribosomal RNA is another popular option, but this RNA has a much higher expression level than most microRNAs, and is often found as a PCR contaminant. If working with blood serum or plasma, please note that only circulating RNA are present. In this case the small non-coding RNAs (5S, U6, SNORs etc) are not good candidates for reference genes, since they are most probably not present in the sample.
58
Using stably expressed microRNAs as reference genes offers several advantages such as equal size, extraction efciency and stability, as well as having expression levels within a similar range of the target microRNAs. Several candidates can be found in the literature, including miR-191, miR-103, let-7a, and miR-16. Microarray or qPCR panel screening data may also be used when choosing candidate reference genes. All reference gene candidates should be empirically validated for each study. A number of different software packages exist for evaluating the optimal nature and number of endogenous controls, and for applying multiple endogenous controls for normalizing target expression. One such option is the GenEx software from MulitD, sold through Exiqon with a special application for Exiqon PCR panels. GenEx incorporates both GeNorm and Normnder for nding the optimal reference genes, and is easy and intuitive to use for the actual normalization. An alternative option for normalization of data from panels (proling a high number of microRNAs) is to normalize against the global mean; that is, the average of all expressed microRNAs This can be a good option in samples with a high call-rate (expressed microRNAs), but should be used with caution in samples with low call-rates. It is also not a good option in samples for which the general microRNA expression level is changed. For further information on normalization and references, we recommend our data-analysis guide available online as well as the guide to microRNA normalization from www.genequantication.de.
59
Troubleshooting guide
Problem PCR signal in samples amplied from rststrand synthesis reactions performed without reverse transcriptase PCR signal in no-template PCR reaction Suggestion This typically indicates contamination of the template RNA with genomic DNA. Perform DNase treatment of the RNA sample. If this does not solve the problem RNA samples or other reagent may be contaminated with PCR products.
This typically indicates contamination of the cDNA template or PCR reagents with amplied PCR product. Exposing the reactions to elevated temperatures (i.e. room temperature) during any part of the protocol increases the risk of background signals. It is important that the reagents and assembled reactions are kept cool (on ice or 4C) at all times (see Tip 4, p. 53 for details).
Generated signals are weak
On some real-time PCR cyclers, gain-settings are adjustable. Make sure the gain settings of your real-time PCR cycler have been set to accommodate the signals generated from the specic assay. RNA samples may contain PCR inhibitors. Further purication or an alternative RNA extraction method may be necessary. Check positive controls. Conrm that you have a PCR product by running an aliquot of your PCR reaction on an agarose gel. Check that the lter in the real-time PCR cycler was set to either SYBR Green or FAM/FITC. Check that the optical read is at the correct step of the real-time PCR cycles. Adjust the baseline in the real-time PCR cycler software.
No uorescent signal is detected during the PCR No uorescent signal detected during the PCR, but a PCR amplicon can be detected by agarose gel electrophoresis
60
FAQs
What kind of real-time PCR instruments is miRCURY LNA Universal RT microRNA PCR compatible with? miRCURY LNA Universal RT microRNA PCR is compatible with all instruments capable of reading green uorophores such as uorescein/FITC/FAM and SYBR Green. The system has been tested and found to work on real-time PCR instruments from several leading suppliers of this type of instrument. What kind of settings should I use on my real-time PCR instrument? If your real-time PCR instrument supports uorophores such as uorescein/FITC/FAM or SYBR Green your instrument must be set to detect these uorophores. Is miRCURY LNA Universal RT microRNA PCR compatible with other SYBR Green master mixes? We do not recommend using other SYBR Green master mixes for real-time PCR analysis with the LNA PCR primer sets or Ready-to-Use panels. The primer sets have been optimized and validated using the miRCURY LNA ExiLENT SYBR Green master mix and the performance of the primer sets will be compromised by using a different master mix (which may contain different salt and/or enzyme concentrations). My RNA is already enriched for microRNA, how much should I use in the real-time PCR experiments? miRCURY LNA Universal RT microRNA PCR is developed for use on total RNA and we do not recommend enriching for small RNAs. Samples of enriched microRNAs are difcult to quantitate accurately making it very tricky to ensure the same amount of sample is added to each reaction. If necessary, a total RNA equivalent should be used for the enriched sample, e.g. use a proportional amount of enriched sample resulting from 20 ng of total RNA. It may be necessary to try a couple of different amounts of enriched sample to ensure that the results fall within the linear range of the assay.
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What is the recommended experimental set up for Universal RT microRNA real-time PCR? It is generally accepted that the reverse transcription (RT) reaction gives rise to more variation than the PCR reaction. It is therefore advisable to perform replicate RT reactions, ideally 3 separate reactions with 1-2 PCR reactions for each RT. It is further recommended to always include at least three biological replicates (separate RNA extractions) of each sample type in order to allow statistical analysis of the results. If small changes in microRNA expression are expected, it may be necessary to include more replicates to ensure a signi cant result. In general it is recommended that replicates should be included at any stage during sample procurement, processing, RNA isolation, etc. that could give rise to variation between samples. A tech note on guidelines for setting up microRNA qPCR experiments can be downloaded at www.exiqon.com/miRNA-qPCR-guidelines Additional information is available at www.exiqon.com/mirna-pcr
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Related products
Exiqon offers a broad variety of tools enabling new discoveries concerning the expression, function and spatial distribution of microRNAs: miRCURY LNA microRNA Array, microarray kit Pre-printed miRCURY LNA microRNA Array microarray slides, available for hsa, mmu & rno and other species. Kit includes hybridization and wash buffers as well as synthetic spike-in microRNAs. miRCURY LNA microRNA Power labeling kit For uorescent labeling of microRNAs from total RNA samples ready for array hybridization. miRCURY LNA microRNA Array, ready-to-spot probe set Ready-to-spot oligos for direct printing of arrays, or coupling in bead-based applications. miRCURY LNA microRNA Detection Probes For in situ hybridization and northern blotting of all annotated microRNAs. miRCURY LNA microRNA ISH Optimization kit (FFPE) Complete kit with control probes and hybridization buffer for easy set up of microRNA
in situ hybridization.
miRCURY LNA microRNA Inhibitors, Power Inhibitors and Family Inhibitors Unravel the function of microRNAs by microRNA inhibition. Sophisticated LNA design ensures potent inhibition of all microRNAs regardless of their GC content. Chemically modied, highly stable Power Inhibitors for unrivalled potency. Available for in vitro and in vivo studies. miRCURY LNA microRNA Inhibitor Library For genome-wide high throughput screening of microRNA function. miRCURY LNA Target Site Blockers For inhibition of microRNA binding to a specic mRNA target.
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Notice to purchaser Exiqon, LNA and miRCURY are registered trademarks of Exiqon A/S, Vedbaek, Denmark. SYBR Green is a licensed trademark of Invitrogen. All other trademarks are the property of their respective owners. Locked-nucleic Acids (LNAs) are protected by US Pat No. 6,268,490, US Pat No. 6,770,748, US Pat No. 6,639,059, US Pat No. 6,734,291 and other applications and patents owned or licensed by Exiqon A/S. Products are provided to buyers for research use only. The products in their original or any modi ed form may be used only for the buyers internal research purposes and not for commercial, diagnostic, therapeutic, or other use, including contract research. The buyer may not provide products to third parties in their original or any modi ed form. The purchase of products does not include or carry an implied right or license for the buyer to use such products in their original or any modi ed form in the provision of services to third parties, and a license must be obtained directly from Exiqon A/S for such uses. Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US: 5,994,056 and 6,171,785. The purchase of this product includes a limited, non transferable immunity from suit under the foregoing patent claims for using only this amount of product solely in Contract Research, including reporting results of purchasers activities for a fee or other commercial consideration, and also for the purchasers own internal research. No right under any other patent claim is conveyed expressly, by implication, or by estoppel. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. Furthermore, this product is provided under an agreement between Molecular Probes, Inc., a wholly owned subsidiary of Invitrogen Corporation, and EXIQON and the manufacture, use, sale or import of this product is subject to one or more U.S. Patents and corresponding international equivalents. The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer, where such research does not include testing, analysis or screening services for any third party in return for compensation on a per test basis. The buyer cannot sell or otherwise transfer (a) this product (b) its components or (c) materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes. Commercial Purposes means any activity by a party for consideration and may include, but is not limited to: (1) use of the product or its components in manufacturing; (2) use of the product or its components to provide a service, information, or data; (3) use of the product or its components for therapeutic, diagnostic or prophylactic purposes; or (4) resale of the product or its components, whether or not such product or its components are resold for use in research. For information on purchasing a license to this product for purposes other than research, contact Molecular Probes, Inc., Business Development, 29851 Willow Creek Road, Eugene, OR 97402. Tel: (541) 4658300, Fax: (541) 335-0354. Further, the purchase of this product includes a limited, non-transferable license under speci c claims of U.S. Patent Nos. 6,174,670 and 6,569,627, owned by the University of Utah Research Foundation and licensed to Roche Diagnostics GmbH and Idaho Technology, Inc., to use only the enclosed amount of product according to the speci ed protocols. No right is conveyed, expressly, by implication, or by estoppel, to use any instrument or system under any claim of U.S. Patent Nos. 6,174,670 and 6,569,627, other than for the amount of product contained herein. For life science research use only. Not for use in diagnostic procedures.
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Notes
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Outside North America Exiqon A/S Skelstedet 16 DK-2950 Vedbaek Denmark Phone +45 45 660 888 Fax +45 45 661 888 North America Exiqon Inc. 12 Gill Street, Suite 1650 Woburn, MA 01801 United States Phone (781) 376 4150 Fax (781) 376 4152 exiqon.com
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Expression Analysis

s, sample id u ic For bio se spec u e s a e pl l at manua a-pcr m/mirn o c . n o exiq

miRCURY LNA Universal RT microRNA PCR

miRCURY LNA Universal RT microRNA PCR Instruction Manual

miRCURY LNA Universal RT microRNA PCR Instruction Manual

Universal cDNA synthesis kit II Description page 4

RNA Spike-in kit (optional)

Description page 6, protocol page 20

Description page 9, protocol page 43

Description page 7, protocol page 29+36

miRCURY LNA Universal RT microRNA PCR Instruction Manual

miRCURY LNA Universal RT microRNA PCR Instruction Manual

2 x 1.25 ml, 2x concentrated 2 x 1.25 ml 500 L, 10x concentrated

2 x 10 ml, 2x concentrated 1 x 20 ml 500 L, 100x concentrated

miRCURY LNA Universal RT microRNA PCR Instruction Manual

II. Primer Sets and Panels

miRCURY LNA Universal RT microRNA PCR Instruction Manual

5 RNA Spike-in control primer sets 1 blank well

5 RNA Spike-in control primer sets 1 blank well

miRCURY LNA Universal RT microRNA PCR Instruction Manual

miRCURY LNA Universal RT microRNA PCR Instruction Manual

Please go to www.exiqon.com/pick-and-mix to congure a Pick-&-Mix microRNA PCR Panel.

miRCURY LNA Universal RT microRNA PCR Instruction Manual

Additional required materials

miRCURY LNA Universal RT microRNA PCR Instruction Manual

Recommended accompanying products

miRCURY LNA Universal RT microRNA PCR Instruction Manual

Step 1: First-strand synthesis (RT)

Step 2: Real-time PCR amplification

miRCURY LNA Universal RT microRNA PCR Instruction Manual

miRCURY LNA Universal RT microRNA PCR Instruction Manual

Table 1. Example of inter-plate calibration (IPC)

Plate 2 20.93 19.70 19.81 -0.11 21.04

Plate 3 21.34 20.00 19.81 0.19 21.15

miRCURY LNA Universal RT microRNA PCR Instruction Manual

miRCURY LNA Universal RT microRNA PCR Instruction Manual

Figure 3. Experimental design

Sold as separate Spike-in kit (203203)

Comes with Universal cDNA synthesis kit II cel-miR-39-3p UniSp6

Included in all panel products

Assays available as individual assays or in ready-to-use panels miR-A miR-X

UniSp2 Sample n Sample n+1 NTC

Sample preparation evaluation

cDNA synthesis evaluation

Inter plate Calibration

miRCURY LNA Universal RT microRNA PCR Instruction Manual

Before starting the Experiment

miRCURY LNA Universal RT microRNA PCR Instruction Manual

miRCURY LNA Universal RT microRNA PCR Instruction Manual

Figure 4. Protocol overview

Use standard printed manual or download from www.exiqon.com/pcr-manual

Use biofluids manual from www.exiqon.com/pcr-manual-biofluids

miRCURY LNA Universal RT microRNA PCR Instruction Manual

Protocol A - Individual assays

Protocol Individual assays 20

miRCURY LNA Universal RT microRNA PCR Instruction Manual

Protocol Individual assays 21

miRCURY LNA Universal RT microRNA PCR Instruction Manual

Workow for individual primer sets (per sample)

Phase I: Prepare RNA sample See page 52 for recommendations

Phase II: cDNA synthesis See protocol page 23.

Protocol Individual assays

LNATM primer sets

Relative expression (log2)