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

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m (→‎Plate Preparation)
(→‎RT-PCR Reaction: added details about RT Reaction)
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===RT-PCR Reaction===
 
===RT-PCR Reaction===
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#Use 8 uL of RNA per RT reaction.
+
#Book PCR Machine for 2h
−
#Use Superscript RT-PCR kit from Invitrogen, following manufacturers instructions
+
#Thaw, mix and quickly spin RNA, dNTP mix, random hexamers, 10X RT buffer, 25 mM MgCl2 water
−
#Store cDNA at -20 until use
+
#Combine the following:
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#Typically dilute 5X in water (adjust for higher or lower expressing transcripts)
+
##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===

Revision as of 11:47, 9 July 2009

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 5 uM mixture of fwd and rev and then make a working solution of 0.4 uL + 4.6 uL water per reaction)
  • 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. 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
  3. Combine the following:
    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
  2. Prepare primer mix from 5 uM primer pair stocks. Per reaction add 0.4 uL primers + 4.6 uL water.
  3. Get 96 well block and keep on rack. Do not touch bottom of plate.
  4. Add 5 uL template per well.
  5. Add 5 uL primer per well.
  6. Add 10 uL per row into 8 wells of a PCR strip plus an extra 10-20 uL
  7. Using a multichannel pipettor, add 10 uL Master mix to each well
  8. Start qPCR Machine using the machine in the Lin Lab or the Saltiel Lab machine

References (Saltiel Lab)

<pubmed>18829989</pubmed> <pubmed>17008399</pubmed> <pubmed>17200717</pubmed> <pubmed>17192460</pubmed> <pubmed>16926380</pubmed>

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