Richard Lab:mRNA quantification

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(RNA extraction)
Current revision (17:59, 29 October 2011) (view source)
(Reverse Transcription)
 
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This page is for mRNA quantification;
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This page is for mRNA quantification using qPCR;
===RNA extraction===
===RNA extraction===
1.Use a kit or take a look at [[RNA extraction|this hub page]]<br>
1.Use a kit or take a look at [[RNA extraction|this hub page]]<br>
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2.Be really carefull when you do this, use DEPC water and some kind of RNAase degrading spray.<br>
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2.Be really carefull when you do this, use DEPC water and some kind of RNAase degrading spray.<br
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3.Typical concentrations after extraction are 500-1000ng/μL.
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You can use a a reverse transcription PCR for this.<br>
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===Reverse Transcription===
===Reverse Transcription===
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1.Prepare the foloowing reaction mix (in this order):
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You could just run RT-qPCR or you can do a two step procedure at use this protocol
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::*2μL Nuclease free water
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1.Dilute the necessary reagents.
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::*1μL Random hexamer mix (60μM)
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::*RNA Diluted to 5ng/μL
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::**Typically a 1:100 dilution
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::*Gene-specific primer mix diluted to 10μM
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::**e.g. If using three primers at 100μM each add 10μL of each primer to 70μL water.
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::**Only the reverse primer is necessary (in the opposite direction of the promoter) 
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2.Prepare the following reaction mix (in this order):
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::*2μL DEPC water
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::*1μL random hexamer mix (60μM)
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::**Alternately 1μL gene specific primer mix (10mM each)
::*5μL AMV reaction mix
::*5μL AMV reaction mix
::*1μL RNA from extraction (500-1000ng/μL)
::*1μL RNA from extraction (500-1000ng/μL)
::*1μL AMV reverse transcriptase
::*1μL AMV reverse transcriptase
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2.Incubate at 25°C for 5 min.  
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3.Incubate at 25°C for 5 min.  
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3.Incubate at 42°C for 1 hour.
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4.Incubate at 42°C for 1 hour.
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4.Inactivate enzyme at 80°C for 5 minutes.
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5.Inactivate enzyme at 80°C for 5 minutes.
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5.Add 20μL nuclease free water.
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6.Add 20μL nuclease free water.
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6.Store at -20°C.
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7.Store at -20°C.

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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