Richard Lab:mRNA quantification: Difference between revisions
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This page is for mRNA quantification; | 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> | ||
2.Be really carefull when you do this, use DEPC water and some kind of RNAase degrading spray.<br | 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=== | ===Reverse Transcription=== | ||
1.Prepare the | You could just run RT-qPCR or you can do a two step procedure at use this protocol | ||
::*2μL | 1.Dilute the necessary reagents. | ||
::*1μL | ::*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 | ::*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 | ||
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. | |||
Revision as of 14:26, 29 October 2011
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)
- RNA Diluted to 5ng/μL
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
- DEPC water to make a total of 8μL at this point.
- 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.
