Real Time PCR From Cell Culture: Difference between revisions

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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 0.22 uM mixture of fwd and rev)
*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===
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).
#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.
#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
#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.


RT-PCR Reaction
===Plate Preparation===
1.Use 8 uL of RNA per RT reaction.
#Book 3h on qPCR machine at http://felix2.lsi.umich.edu/ORS, username '''and''' password is davebrid
2.Use Superscript RT-PCR kit from Invitrogen, following manufacturers instructions
#Get 96 or 384 well block and keep on rack. Do not touch bottom of plate.
3.Store cDNA at -20 until use
#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


Plate Preparation
==Calculations==
1.Prepare dilutions of primers. Need 9 uL per well. Book 3h on qPCR machine
see http://www.ncbi.nlm.nih.gov/pmc/articles/PMC55695 for considerations on calculations
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)
==References (Saltiel Lab)==
<pubmed>18829989</pubmed>
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

  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

  1. Book PCR Machine for 2h
  2. 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
  3. Combine the following in a PCR tube:
    1. 8 uL RNA
    2. 1 uL dNTP mix
    3. 1 uL Random Hexamers
  4. Put in PCR machine and run program RT 65C (takes 5 min)
  5. In a separate tube add in the following order (for 10 reactions):
    1. 20 uL 10X RT buffer
    2. 40 uL 25 mM MgCl2
    3. 20 uL 0.1M DTT
    4. 10 uL RNAse Out
  6. Add 9 uL of the reaction mix to each primer/RNA mixture from previous step, mix and quickly centrifuge
  7. Sit on the bench for 2 min
  8. Add 1 uL SuperScript II RT to each tube
  9. Put in PCR machine and run program RT Reaction. After run (75 min) place on ice.
  10. Add 1 uL RNase H and put in PCR machine and run program RT 37. Takes 20 min.
  11. Store cDNA at -20 until use.

Plate Preparation

  1. 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.
  3. Add 5 uL template per well. If using a 384 well plate use 2.5uL
  4. Add 5 uL primer per well. If using a 384 well plate use 2.5uL
  5. Add 10 uL per row into 8 wells of a PCR strip plus an extra 10-20 uL
  6. Using a multichannel pipettor, add 10 uL Master mix to each well. If using a 384 well plate use 5uL
  7. 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

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