Difference between revisions of "Real Time PCR From Cell Culture"

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(added reference for calculating amounts from Ct)
 
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__NOTOC__
 
==Real Time qPCR==
 
==Real Time qPCR==
 
===Materials===
 
===Materials===
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*SyberGreen PCR Master Mix Applied Biosystems
 
*SyberGreen PCR Master Mix Applied Biosystems
 
*96 well qPCR plate
 
*96 well qPCR plate
−
*Primers (Dilute to 0.22 uM mixture of fwd and rev)
+
*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).
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#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===
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#Use 8 uL of RNA per RT reaction.
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#Book PCR Machine for 2h
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#Use Superscript RT-PCR kit from Invitrogen, following manufacturers instructions
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#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
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#Store cDNA at -20 until use
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#Combine the following in a PCR tube:
−
#Typically dilute 5X in water (adjust for higher or lower expressing transcripts)
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##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/index.php?UID=2fd952b00de25
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#Book 3h on qPCR machine at http://felix2.lsi.umich.edu/ORS, username '''and''' password is davebrid
−
#Prepare primer mix from 5 uM primer pair stocks by adding 0.4 uL primers + 4.6 uL water per reaction
+
#Get 96 or 384 well block and keep on rack.  Do not touch bottom of plate.
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#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)==
−
<pubmed>18829989</pubmed>
+
PMID 18829989
−
<pubmed>17008399</pubmed>
+
PMID 17008399
−
<pubmed>17200717</pubmed>
+
PMID 17200717
−
<pubmed>17192460</pubmed>
+
PMID 17192460
−
<pubmed>16926380</pubmed>
+
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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