V7.

6 Step-by-Step RNA extraction protocol for cells with RNeasy Mini kit and RNeasy plus kit (Qiagen). 1) If using trypsin to collect cells from plates: Aspirate the cell culture medium> wash cells on plate with PBS> aspirate PBS> add trypsin, incubate in 37oC> after cells detach from the plate> add medium twice the amount of the added trypsin to inactivate the trypsin> transfer the supernatant with the cells to an RNase free sterile tube> Centrifuge in 1100 rpm for 7 minutes> Completely aspirate the cell culture medium. * If using frozen cells: Freeze only the cells pellet without any medium or buffer and store in -80oC. 2) Determine the number of cells. 3) Add 10l -mercaptoethanol (-ME) per 1ml buffer RLT. When working with -ME use fume hood. RLT+-ME can be stored at R.T for 1 month. 4) Add RLT+-ME to the cells pellet. Vortex and pipette to mix. * Cells can be also harvested directly from the dish, after RLT addition (without trypsin). Cells grown in flask, should always be trypsinized and harvested into a tube before RLT addition.

5) Homogenize the lysate. Pass the lysate at least 5 times through a syringe and a 2025 gouge needle. At this point the lysate can be stored in 80oC and processed later. 6) If using the Rneasy plus mini kit (Qiagen): transfer the homogenized lysate to a gDNA eliminator spin column. Centrifuge for 30s at 8000g 10,000 RPM. Discard the column and save the flow- through. (use this step instead of step 10). 7) Add 1 volume of ethanol 70% to 1 volume of homogenized lysate. Mix well by pipetting. DO NOT Centrifuge. * Use 100% ethanol diluted with Ultra pure water. 8) Transfer up to 700 l of the sample to an RNeasy spin column. Place the column inside a 2ml tube (supplied with the kit). 9) Centrifuge for 1min at full speed (8000g 10,000 RPM). Discard flow. If using -ME place the column in a new tube in a fume hood, discard the old one. If the sample volume is larger than 700 l, centrifuge successive aliquots in the same RNeasy spin column. Discard the flow-through after each centrifugation.

10) If performing optional on-column DNase digestion, use Qiagen RNase free DNase set kit ( use this step if not using step 6). Proceed to Appendix A 11) Add 700 l RW1 to the column and centrifuge. Discard the flow-through. Use the collection tube from step 7. * If using Dnase digestion, skip this step. 12) Add 500 l RPE to the column and centrifuge. Discard the flow-through. Use the collection tube from step 7. * Before using the buffer RPE for the first time, add 100% ethanol as indicated on the bottle and mark the bottle. 13) Add 500 l RPE to the column and centrifuge for 2 min. Discard the flowthrough. Use the collection tube from step 7. 14) Place the column in a new 2 ml tube and centrifuge for 1min. 15) Place the column in a new 1.5 ml tube, add 40l RNase free water (supplied). Insert the water directly on top of the membrane inside the tube. * If using small amount of cells, add 20l, or less. 16) Leave the tube for 2 min at R.T. 17) Centrifuge for 1 min . The flow-through now contains the RNA. 18) Repeat steps 15-17 again to increase the RNA amount. Use the same tube from step 15. * When performing step 16 again with additional 40 l of RNase-free water, the RNA amount will be higher, but the concentration lower. If using the elution from step16 the RNA yield will be 1530% less than that obtained using a second volume of RNase-free water, but the final RNA concentration will be higher. 19) From this step onwards always keep the produced RNA on ice. Or store in -80oC 20) Test the produced RNA in Nano-drop and in agarose gel. * When testing the product in the Nano-drop the ratio of 260/280 should be 2.0 and higher * If the RNA is not broken, only 2 bands will appear in the gel. Intact total RNA run on a 1% agarose gel will have sharp, clear 28S and 18S rRNA bands (eukaryotic samples). The 28S rRNA band should be approximately twice as intense as the 18S rRNA band. This 2:1 ratio (28S:18S) is a good indication that the RNA is completely intact. Partially degraded RNA will have a smeared appearance, will lack the sharp rRNA bands, or will not exhibit the 2:1 ratio of high quality RNA. Completely degraded RNA will appear as a very low molecular weight smear.

Appendix A - DNase digestion Use "RNase free DNase set" kit from Qiagen 1) Add 350l RW1 to the RNeasy column. Use the sample column from step 8 in the RNeasy section. 2) Centrifuge. Discard flow 3) Add 10 l DNase1 to 70 l buffer RDD. Mix gently by flicking the tube. DO Not Vortex. * Before using Dnase1for the first time, inject 550 l of RNase free water (supplied) into the vial with RNase free syringe and needle. Mix gently, DO NOT Vortex. Divide into single use aliquots of 10 l and store for up to 9 months in -20oC. Thawed aliquots can be stored for up to 6 weeks in 4oC 4) Add 80 l of the solution from step 3 directly to the column membrane. Be sure to add all of the solution directly to the membrane. 5) Leave for 15 min in R.T 6) Add 350 l RW1 to the column. Discard flow 7) Centrifuge. 8) Continue to step 11 in the RNeasy section.