Richard Lab:mRNA quantification

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Current revision (17:59, 29 October 2011) (view source)
(Reverse Transcription)
 
Line 18: Line 18:
::**Alternately 1μL gene specific primer mix (10mM each)
::**Alternately 1μL gene specific primer mix (10mM each)
::*5μL AMV reaction mix
::*5μL AMV reaction mix
-
::*DEPC water to make a total of 8μL at this point.
 
::*1μL RNA from extraction (500-1000ng/μL)
::*1μL RNA from extraction (500-1000ng/μL)
::*1μL AMV reverse transcriptase
::*1μL AMV reverse transcriptase

Current revision

This page is for mRNA quantification using qPCR;

RNA extraction

1.Use a kit or take a look at this hub page
2.Be really carefull when you do this, use DEPC water and some kind of RNAase degrading spray.<br 3.Typical concentrations after extraction are 500-1000ng/μL.

Reverse Transcription

You could just run RT-qPCR or you can do a two step procedure at use this protocol 1.Dilute the necessary reagents.

  • RNA Diluted to 5ng/μL
    • Typically a 1:100 dilution
  • Gene-specific primer mix diluted to 10μM
    • e.g. If using three primers at 100μM each add 10μL of each primer to 70μL water.
    • Only the reverse primer is necessary (in the opposite direction of the promoter)

2.Prepare the following reaction mix (in this order):

  • 2μL DEPC water
  • 1μL random hexamer mix (60μM)
    • Alternately 1μL gene specific primer mix (10mM each)
  • 5μL AMV reaction mix
  • 1μL RNA from extraction (500-1000ng/μL)
  • 1μL AMV reverse transcriptase

3.Incubate at 25°C for 5 min. 4.Incubate at 42°C for 1 hour. 5.Inactivate enzyme at 80°C for 5 minutes. 6.Add 20μL nuclease free water. 7.Store at -20°C.

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