906
edits
Changes
added reference for calculating amounts from Ct
__NOTOC__
==Real Time qPCR==
===Materials===
*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===
#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).
#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 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 of RNA per ##1 uL dNTP mix##1 uL Random Hexamers#Put in PCR machine and run program '''RT reaction.65C''' (takes 5 min)#Use Superscript In a separate tube add in the following order (for 10 reactions):##20 uL 10X RT-PCR kit 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 Invitrogenprevious step, following manufacturers instructionsmix 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===
#Prepare dilutions of primers. Need 9 uL per well. Book 3h on qPCR machine at http://felix2.lsi.umich.edu/ORS, username '''and''' password is davebrid#Get 96 or 384 well block and keep on rack. Do not touch bottom of plate.#Add 1 5 uL template per well. If using a 384 well plate use 2.5uL#Add 9 5 uL primer per well. If using a 384 well plate use 2.5uL#Using Add 10 uL per row into 8 wells of a PCR strip and plus an extra 10-20 uL#Using a multichannel pipettor, add 10 uL Master mix to each well. If using a 384 well plate use 5uL#Start qPCR Machine using the machine in the [[qPCR - Lin Lab|Lin Lab]] or the [[qPRC - Saltiel Lab|Saltiel Lab]] machine ==Calculations==see http://www.ncbi.nlm.nih.gov/pmc/articles/PMC55695 for considerations on calculations
==References (Saltiel Lab)==
