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 1.Use RNEasy kit with Qiashredder. 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.Use 8 uL of RNA per RT reaction. 2.Use Superscript RT-PCR kit from Invitrogen, following manufacturers instructions 3.Store cDNA at -20 until use

Plate Preparation 1.Prepare dilutions of primers. Need 9 uL per well. Book 3h on qPCR machine 2.Get 96 well block and keep on rack. Do not touch bottom of plate. 3.Add 1 uL template per well. 4.Add 9 uL primer per well 5.Using PCR strip and multichannel pipettor, add 10 uL Master mix to each well

References (Saltiel Lab) <pubmed>18829989</pubmed>