Real Time PCR From Cell Culture: Difference between revisions
Jump to navigation
Jump to search
Davebridges (talk | contribs) added reference for calculating amounts from Ct |
|||
| (10 intermediate revisions by the same user not shown) | |||
| Line 1: | Line 1: | ||
__NOTOC__ | |||
==Real Time qPCR== | ==Real Time qPCR== | ||
===Materials=== | ===Materials=== | ||
| Line 6: | Line 7: | ||
*SyberGreen PCR Master Mix Applied Biosystems | *SyberGreen PCR Master Mix Applied Biosystems | ||
*96 well qPCR plate | *96 well qPCR plate | ||
*Primers (Dilute to | *Primers (Dilute to 0.4 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 | *Generate primers using http://pga.mgh.harvard.edu/primerbank/index.html | ||
==Protocol== | ==Protocol== | ||
===RNA Extraction=== | ===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). | #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. | #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 | #Store at -20 until RT reaction | ||
===RT-PCR 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 | ||
#Store cDNA at -20 until use | #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=== | ===Plate Preparation=== | ||
#Book 3h on qPCR machine at http://felix2.lsi.umich.edu/ORS | #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. | |||
#Get 96 well block and keep on rack. Do not touch bottom of plate. | #Add 5 uL template per well. If using a 384 well plate use 2.5uL | ||
#Add 5 uL template per well. | #Add 5 uL primer per well. If using a 384 well plate use 2.5uL | ||
#Add 5 uL primer per well | |||
#Add 10 uL per row into 8 wells of a PCR strip plus an extra 10-20 uL | #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 | #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)== | ==References (Saltiel Lab)== | ||
PMID 18829989 | |||
PMID 17008399 | |||
PMID 17200717 | |||
PMID 17192460 | |||
PMID 16926380 | |||
[[Category:Transcription]] | |||
[[Category:Expression]] | |||
[[Category:RNA]] | |||
[[Category:qPCR]] | |||
Latest revision as of 00:42, 29 March 2011
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 0.4 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
- 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
- Get 96 or 384 well block and keep on rack. Do not touch bottom of plate.
- Add 5 uL template per well. If using a 384 well plate use 2.5uL
- Add 5 uL primer per well. If using a 384 well plate use 2.5uL
- 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. If using a 384 well plate use 5uL
- Start qPCR Machine using the machine in the Lin Lab or the Saltiel Lab machine
Calculations
see http://www.ncbi.nlm.nih.gov/pmc/articles/PMC55695 for considerations on calculations
References (Saltiel Lab)
PMID 18829989 PMID 17008399 PMID 17200717 PMID 17192460 PMID 16926380