Real Time PCR From Cell Culture

Real Time qPCR

Materials

• cDNA for templates
• Qiashredder and RNEasy kits from Qiagen
• Superscript Kit from Invitrogen
• SyberGreen PCR Master Mix Applied Biosystems
• 96 well qPCR plate
• Primers (Dilute to 5 uM mixture of fwd and rev and then make a working solution of 0.4 uL + 4.6 uL water per reaction)
• Generate primers using http://pga.mgh.harvard.edu/primerbank/index.html

Protocol

RNA Extraction

1. Use RNEasy kit with Qiashredder. see Harvesting RNA from Cells grown in monolayer. For a 12 well plate, use 350 uL of RLT (add 10 uL B-ME per 1 mL of RLT buffer).
2. Scrape cells and pass through Qiashredder column. Do the optional DNAse step at step 5 using the RNAse free DNAse from Qiagen.
3. Store at -20 until RT reaction

RT-PCR Reaction

1. Book PCR Machine for 2h
2. Thaw, mix and quickly spin RNA, dNTP mix, random hexamers, 10X RT buffer, 25 mM MgCl2 water
3. Combine the following:
   1. 8 uL RNA
   2. 1 uL dNTP mix
   3. 1 uL Random Hexamers
4. Put in PCR machine and run program RT 65C (takes 5 min)
5. In a separate tube add in the following order (for 10 reactions):
   1. 20 uL 10X RT buffer
   2. 40 uL 25 mM MgCl2
   3. 20 uL 0.1M DTT
   4. 10 uL RNAse Out
6. Add 9 uL of the reaction mix to each primer/RNA mixture from previous step, mix and quickly centrifuge
7. Sit on the bench for 2 min
8. Add 1 uL SuperScript II RT to each tube
9. Put in PCR machine and run program RT Reaction. After run (75 min) place on ice.
10. Add 1 uL RNase H and put in PCR machine and run program RT 37. Takes 20 min.
11. Store cDNA at -20 until use.

Plate Preparation

1. Book 3h on qPCR machine at http://felix2.lsi.umich.edu/ORS
2. Prepare primer mix from 5 uM primer pair stocks. Per reaction add 0.4 uL primers + 4.6 uL water.
3. Get 96 well block and keep on rack. Do not touch bottom of plate.
4. Add 5 uL template per well.
5. Add 5 uL primer per well.
6. Add 10 uL per row into 8 wells of a PCR strip plus an extra 10-20 uL
7. Using a multichannel pipettor, add 10 uL Master mix to each well
8. Start qPCR Machine using the machine in the Lin Lab or the Saltiel Lab machine

References (Saltiel Lab)

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