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 0.22 uM mixture of fwd and rev)
*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===
#Use 8 uL of RNA per RT reaction.
#Use Superscript RT-PCR kit from Invitrogen, following manufacturers instructions
#Store cDNA at -20 until use

===Plate Preparation===
#Prepare dilutions of primers.  Need 9 uL per well.  Book 3h on qPCR machine 
#Get 96 well block and keep on rack.  Do not touch bottom of plate.
#Add 1 uL template per well.  
#Add 9 uL primer per well
#Using PCR strip and multichannel pipettor, add 10 uL Master mix to each well

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