Materials and Methods.

CHAPTER 3
MATERIALS AND METHODS
MATERIALS
Equipments
Instruments used during the present study included- Refrigerated centrifuge
(Sigma 3K-30, Germany), DNA sequencer (3130 XL, Applied Biosystems,
USA), Refrigerated microfuge (Heraeus, Germany), C24KC Incubator
Shaker (Edison, NJ, USA), Thermocycler (ABI Perkin Elmer, USA and
Bio-Rad Research Laboratories, CA, USA), Horizontal Electrophoresis
Unit (Bio-Rad, Research Laboratories, CA, USA), UV Transilluminator
(UVP, USA), Microwave Oven (Whirlpool, India), Magnetic Stirrer
(Biosan, India ), Vortex Shaker CM101 (Remi, India), Autoclave (Microsil,
India), Rotor Plate Centrifuge (Sigma 4-15, Germany), pH Meter (ThermoOrion, USA), Electronic balance (Sartorius, Germany),

Oven (Yorco,

India) and Water purification system (Millipore, France).

Camel populations and collection of blood samples
A total of 6 camel populations were chosen in the present investigation. For
the present study blood samples were collected from their respective
breeding tracts, comprising of Arid, Semi-arid and humid regions. The sites
of blood samples collection are shown in (figure1). About 8 ml of blood
sample was collected from jugular vein of randomly selected camel in
10ml Heparinized vacutainers and the blood was well mixed with heparin
to prevent clotting. The blood samples were transported and stored at 4 oC
prior to isolation of DNA. Essentially Rajasthan, Gujarat and Madhya
Pradesh are predominantly the center of Camel rearing and breeding tracts
existing in India.

27

Materials and Methods.

3.3. DNA Isolation, Storage, Quantification

3.3.1. Isolation of DNA
DNA isolation was carried out by using standard Phenol/chloroform
extraction protocol followed by ethanol precipitation from blood samples.
The RBCs were lysed with lysis buffer then mixed gently and kept in ice
for 10 minutes. The tubes were centrifuged at 12,000 rpm for 10 minutes at
4C in the refrigerated centrifuge. The supernatant was carefully decanted
and the pellet was redissolved and washed three times in lysis buffer. The
pellet was resuspended in 10ml digestion buffer (Sodium chloride 75 mM,
Tris-Cl 1 mM, pH 8.0 and EDTA 0.5 mM). Vortexed gently and added 20%
Sodium Lauryl Sulphate (200 l/sample) and Proteinase K (1 mg/sample)
to it and incubated at 57C overnight in incubator. After incubation,
digested solution was obtained to which equal amount of Tris equilibrated

28

Materials and Methods.

phenol (pH 8.0) was added. This was mixed gently by moving the tubes
gently in 8 fashion for 10 minutes and centrifuge at 12,000 rpm for 10
minutes at 25C. After centrifugation the aqueous phase and organic phase
were separated. The aqueous phase was transferred carefully by Pasteur
pipette to another Oakridge tube without disturbing the interphase. To the
aqueous phase, phenol: chloroform: isoamyl alcohol (25:24:1) was added
and mixed by moving the tubes gently in 8 fashions for 10 minutes at
25C to separate the aqueous phase and organic phase. Again the aqueous
phase was carefully pipetted to another tube without disturbing the
interphase. The aqueous phase was mixed with chloroform: isoamyl
alcohol (24:1). The solution was subjected to centrifugation at 12,000 rpm
for 10 minutes at 25C and aqueous phase was carefully transferred to glass
culture tube. The DNA was precipitated by adding 1/10 volume of sodium
acetate (3 M, pH 5.2) and 2.5 volume of aqueous phase of chilled absolute
alcohol. The DNA was spooled out into eppendorf tubes and washed twice
with 70% ethanol to remove the salt. After washing, the alcohol was
allowed to evaporate and DNA was dissolved in 500 ml Tris EDTA buffer
(Tris 10 mM, EDTA 10mM, pH8.0). Kept out Eppendorf tubes at 65C for
1 hour. The stock DNA was stored at -20C.
Estimation of DNA quantity and purity
DNA quantification was done by diluting genomic DNA in sterile
distilled water in 1:100 ratios and measuring optical density at 260nm and
280nm against a blank (sterile distilled water) using UV-Visible
spectrophotometer. The concentration of unknown double stranded DNA
samples was estimated using the formula:

Quality of Genomic DNA

Quantification of the DNA can be achieved by running the DNA samples
on 0.6 % agarose gel stained with Ethidium bromide (0.5 mg per ml) and
image was captured. Intact genomic DNA band with slight smear was

29

Materials and Methods.

observed in wells. A smeared band towards the bottom of the gel is an
indication of the presence of RNA in the extract. A rough estimate of DNA
content (50ng/l) may be obtained by comparing band intensities of the
DNA extract and the standard by eyes.

30

Materials and Methods.

Figure: Showing quality of genomic DNA which was analyzed on 0.8

% agarose gel electrophoresis

3.1 Tissue Sample Collection

For RNA-Seq data generation, all the five tissues (Heart, Kidney,
testis, skin and lungs) were taken from camelus dromedarius. The fresh
tissues were steeped in RNAlater (Stabilization Reagent) immediately after
collection. RNA Stabilization reagent stabilizes RNA in tissue samples,
and then kept at 80 C and brought to lab and processed immediately for
RNA extraction.
3.2 RNA Isolation
A typical mammalian cell contains about 10 _11g of RNA of which
1-5% is poly A+ RNA. The remaining RNA is mostly rRNA (80-85% of
total) and low-molecular weight RNAs such as tRNA (15-20% of total). To
separate the heterogeneous population of mRNA from the majority of the
RNA found in the cell, affinity binding to oligo (dT) cellulose is used. This
method exploits the major characteristics of mRNA, polyadenylation, to
obtain intact, pure mRNA.
Rnase-free Glassware
Glassware used for RNA work should always be: cleaned with
detergent, thoroughly rinsed, autoclaved, then oven baked at > 210C for

31

Materials and Methods.

atleast three hours before use. Oven- baking will both inactivate
ribonucleases and ensure that no other nucleic acids (such as plasmid
DNA) are left on the surface of the glassware. Do not use any plastic or
glassware without first eliminating possible ribonuclease contamination
from the surface of the skin. Always use proper microbiological aseptic
technique when working with RNA.

Flow Chart of Process: The following figure outlines the FastTrack@

process

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Materials and Methods.

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Materials and Methods.

1) Preparation of lysis Buffer: Add 300l of protein/RNase
Degrader to 15 ml of stock Buffer and use immediately.
2) Frozen tissue samples were taken out from the storage and put it in
a tared (pre-weighed), sterile, 50ml centrifuge tube and added 15 ml
Lysis buffer.
3) Sample was quickly homogenized using a homogenizer for 40 sec.
4) The cell lysate was incubated at 45C for 15-60 minutes. If
insoluble material persists, centrifuge at 4,000x g for 5 minutes at
room temperature and transfer the supernatant to a new Eppendorf
tube.
5) 1 volume of 70% ethanol was added to cleared lysate and mixed it
well by pipetting.
6) Added 700l of the sample into an RNeasy mini spin column
sitting in a 2 ml collection tube.
7) Centrifuged all the samples at 12,000 rpm for 15 sec. Discarded
flow-through and reused the collection tube.
8) Added 700l RW1 buffer onto the RNeasy column, incubated
RNeasy column for 5 min, and centrifuged for 15 sec at maximum
speed. Discarded flow-through and the collection tube.
9) Transferred RNeasy column to a new 2ml collection tube.
10) Added 500l Buffer RPE onto RNeasy column, and centrifuged for
15 sec at maximum speed to wash. Discarded flow-through and
reused the collection tube.
11) Again added 500l Buffer RPE onto RNeasy column, and
centrifuged for 2 min at maximum speed to dry the RNeasy
membrane. Discarded flow-through and the collection tube.
12) Transferred RNeasy column into a new 1.5ml Eppendorf tube
(remove the lid) and pipetted 30l of RNase-free water directly
onto the RNeasy membrane. Centrifuged for 1 min at maximum
speed to elute the RNA.

34

Materials and Methods.

13) Transferred elute to a new Eppendorf tube and stored at -80C in
RNase-free water.
3.3 Quantitation and quality checking of RNA
The mRNA comprises only 1- 3% of total RNA samples. It was not readily
detectable even with the most sensitive of methods. Determining the
integrity of RNA starting materials is a critical step in Transcriptome
analysis. Quantification of RNA was done using Agilent 2100 Bioanalyzer
(Agilent, Foster city, USA) which provided ng RNA/l values. The Agilent
2100 bioanalyzer and associated RNA 6000 Nano and Pico Lab Chip kits
have become the standard in RNA quality assessment and quantitation.
Using electrophoretic separation on micro-fabricated chips, RNA samples
are separated and subsequently detected via laser induced fluorescence
detection. The bioanalyzer software generates an electropherogram and
gel-like image and displays results such as sample concentration and the so
called ribosomal ratio. The electropherogram provides a detailed visual
assessment of the quality of an RNA sample. The clear 28S and 18S rRNA
bands were indicative of intact RNA. 2100 Bioanalyzer was used with each
tissue sample and when the RNA integrity number was found to be less
than 8.5, RNA of that tissue was again isolated and further analyzed for
RNA integrity number. The RNA samples were processed further if the
RNA Integrity Number (RIN) was found to be greater than 8.5, for RNASeq library preparation.
3.4 RNA-Seq library preparation and data generation
DNA library preparation was done by using Standard Illumina kit
facilitated reading both the forward and reverse template strands of each
cluster during one paired-end read. In addition to sequence information,
both reads contain long range positional information, allowing for highly
precise alignment of reads. The paired-end sequencing assay utilized a
combination of cBot (or the Cluster Station) and the paired-end module
followed by paired-end sequencing on the Genome Analyser IIx. The
unique paired-end sequencing protocol allowed us the length of the insert

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Materials and Methods.

(200300 bp), generating high quality and alignable sequence data. A
typical paired-end run could achieve 276 bp reads and up to 40-60 million
reads of the transcriptome data for each of the 5 tissue samples of Camelus
dromedarius. This part of work has been outsourced.
3.5 Transcriptome data analysis
RNA-seq data analysis was done by using different software.
Transcriptome data was subjected to

Alignment against a reference genome (bovine whole genome

sequence: Btau4.0)

Identification of Molecular markers

Gene ontology

3.5.1 Quality control of RNA-Seq data

The reads may look OK in QC analyses of raw reads but some issues only
show up once the reads are aligned like low coverage, homopolymer
biases, experimental artifacts, etc. These unwanted biases can be
introduced during sample extraction process, sequencing technology,
sample preparation protocol and or may be during mapping algorithm.
SAM/BAM files usually contain information from tens to hundreds of
millions of reads. The systematic detection of such biases is a non-trivial
task that is crucial to drive appropriate downstream analyses. RNA-Seq
generated data were subjected for quality scrutiny by using SAMStat
software (Lassmann et al., 2011).
3.5.2 Alignment of RNA-seq data against reference genes
We utilized TopHat software version 1.3.2 for alignment of Transcriptome
data to cattle genome Btau 4.0 downloaded from UCSC Genome browser
(http://genome.ucsc.edu/). 3.5.3 Assembly of RNA-seq data
For assembly of transcripts, Cufflinks software version 1.0.2 was utilized.
RNA-seq read alignments in SAM/BAM format which was used as input
file for Cufflinks and it generates transcripts file.
3.5.4 Extracting gene sequencing and mapping for gene ontology terms

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Materials and Methods.

The sequences of the transcriptome were extracted using the facility of
CLCBio. All the reads were mapped with the GO IDs. The gene names
were mapped using webserver g: Profiler (http://biit.cs.ut.ee/gprofiler/) for
classification into Biological process, Cellular component and Molecular
function. REViGO web server (http://revigo.irb.hr/) was utilized for
summarizing the long lists of Gene Ontology terms associated with these
genes for removal of redundant GO terms.
3.6 Sequence Submission
All the align sequence of camel against reference sequence were extracted
and uploaded in NCBI (National Centre of Biotechnology Information).
3.7 Identification of new molecular markers
3.7.1 SNP identification
RNA-Seq data generated was mapped for each of the tissues of camel
using Illumina GAIIx with cattle genome Btu 4.0. The resultant mapped
files were then merged and utilized for heterozygous SNP calling. For SNP
detection,

CLC

Genomics

Workbench

was

used

which

utilizes

Neighborhood Quality Standard (NQS) algorithm (Altshuler et al., 2000).

The central base quality score of 20 and average surrounding base quality
score of 15 were set to assess the quality of reads at positions for SNP
detection. We utilized the criteria of depth of coverage of ten and the minor
allele frequency of 2 out of 10 reads (minimum allele frequency of 20%)
for the identification of SNPs.
3.7.2 Microsatellite identification
For detection of STRs, novel repeat detection program PHOBOS has been
chosen for the STADEN pipeline since it implements a fast, efficient, and
highly accurate algorithm to scan molecular sequences for tandem repeats.
The minimal seed size is 5 repeat units for tandem repeats and for repeats
with longer units at least 2 exact units must be present. The Mismatch score
of -5, Indel score of -5 and recursion depth of 5 for high quality alignment
and perfection range set for 80 to 100%.
3.8 Validation of genetic marker

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Materials and Methods.

What about PCR amplification, cocktail preparation, genotyping by DNA
sequencer and data extraction

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