Difference between revisions of "Real Time PCR From Cell Culture"

Revision as of 18:49, 21 July 2009

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. All reagents are in the small red invitrogen box
3. Combine the following in a PCR tube:
   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, username and password is davebrid
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)

<pubmed>18829989</pubmed> <pubmed>17008399</pubmed> <pubmed>17200717</pubmed> <pubmed>17192460</pubmed> <pubmed>16926380</pubmed>