Real Time PCR From Cell Culture
From Bridges Lab Protocols
Real Time qPCR
- 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 0.4 uM mixture of fwd and rev. From 100 uM stocks this is 4uL Forward Primer, 4 uL Reverse Primer and 992 uL Water)
- Generate primers using http://pga.mgh.harvard.edu/primerbank/index.html
- Use RNEasy kit with Qiashredder. see Harvesting RNA from Cells grown in monolayer or Preparation_of_RNA_Samples_from_Mouse_Tissues. For a 12 well plate, use 350 uL of RLT (add 10 uL B-ME per 1 mL of RLT buffer).
- Scrape cells and pass through Qiashredder column. Do the optional DNAse step at step 5 using the RNAse free DNAse from Qiagen.
- Store at -20 until RT reaction
- Book PCR Machine for 2h
- Thaw, mix and quickly spin RNA, dNTP mix, random hexamers, 10X RT buffer, 25 mM MgCl2 water. All reagents are in the small red invitrogen box
- Combine the following in a PCR tube:
- 8 uL RNA
- 1 uL dNTP mix
- 1 uL Random Hexamers
- Put in PCR machine and run program RT 65C (takes 5 min)
- In a separate tube add in the following order (for 10 reactions):
- 20 uL 10X RT buffer
- 40 uL 25 mM MgCl2
- 20 uL 0.1M DTT
- 10 uL RNAse Out
- Add 9 uL of the reaction mix to each primer/RNA mixture from previous step, mix and quickly centrifuge
- Sit on the bench for 2 min
- Add 1 uL SuperScript II RT to each tube
- Put in PCR machine and run program RT Reaction. After run (75 min) place on ice.
- Add 1 uL RNase H and put in PCR machine and run program RT 37. Takes 20 min.
- Store cDNA at -20 until use.
- Book 3h on qPCR machine at http://felix2.lsi.umich.edu/ORS, username and password is davebrid
- Get 96 or 384 well block and keep on rack. Do not touch bottom of plate.
- Add 5 uL template per well. If using a 384 well plate use 2.5uL
- Add 5 uL primer per well. If using a 384 well plate use 2.5uL
- Add 10 uL per row into 8 wells of a PCR strip plus an extra 10-20 uL
- Using a multichannel pipettor, add 10 uL Master mix to each well. If using a 384 well plate use 5uL
- Start qPCR Machine using the machine in the Lin Lab or the Saltiel Lab machine
see http://www.ncbi.nlm.nih.gov/pmc/articles/PMC55695 for considerations on calculations