View source for 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===
#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).
#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

===RT-PCR 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.

===Plate Preparation===
#Book 3h on qPCR machine at http://felix2.lsi.umich.edu/ORS, username '''and''' password is davebrid
#Prepare primer mix from 5 uM primer pair stocks.  Per reaction add 0.4 uL primers + 4.6 uL water.
#Get 96 well block and keep on rack.  Do not touch bottom of plate.
#Add 5 uL template per well.  
#Add 5 uL primer per well.
#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
#Start qPCR Machine using the machine in the [[qPCR - Lin Lab|Lin Lab]] or the [[qPRC - Saltiel Lab|Saltiel Lab]] machine

==References (Saltiel Lab)==
<pubmed>18829989</pubmed>
<pubmed>17008399</pubmed>
<pubmed>17200717</pubmed>
<pubmed>17192460</pubmed>
<pubmed>16926380</pubmed>