Difference between revisions of "Real Time PCR From Cell Culture"
From Bridges Lab Protocols
Davebridges (Talk | contribs) (Created page with '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 plat...') |
Davebridges (Talk | contribs) (added reference for calculating amounts from Ct) |
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| − | Real Time qPCR | + | __NOTOC__ |
| − | Materials | + | ==Real Time qPCR== |
| − | cDNA for templates | + | ===Materials=== |
| − | Qiashredder and RNEasy kits from Qiagen | + | *cDNA for templates |
| − | Superscript Kit from Invitrogen | + | *Qiashredder and RNEasy kits from Qiagen |
| − | SyberGreen PCR Master Mix Applied Biosystems | + | *Superscript Kit from Invitrogen |
| − | 96 well qPCR plate | + | *SyberGreen PCR Master Mix Applied Biosystems |
| − | Primers (Dilute to 0. | + | *96 well qPCR plate |
| − | Generate primers using http://pga.mgh.harvard.edu/primerbank/index.html | + | *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 | + | ==Protocol== |
| − | RNA Extraction | + | ===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 | |
| − | 2. | + | #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 [[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
