906
edits
Changes
added reference for calculating amounts from Ct
__NOTOC__==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 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
==Protocol=====RNA Extraction===1.#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).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===
#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.
==References (Saltiel Lab)==<pubmed>PMID 18829989</pubmed>PMID 17008399PMID 17200717PMID 17192460PMID 16926380 [[Category:Transcription]][[Category:Expression]][[Category:RNA]][[Category:qPCR]]
