QRT-PCR: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
No edit summary |
(→Notes) |
||
Line 15: | Line 15: | ||
*#This approach could reduce experimental variability from RNA degradation. | *#This approach could reduce experimental variability from RNA degradation. | ||
*To make total cDNA | *To make total cDNA | ||
*# | *#Use a polyT primer (most but not all eukaryotic mRNA) or random decamers (prokaryotic mRNA). | ||
*#Random decamers give longer cDNAs on average than random hexamer primers. | *#Random decamers give longer cDNAs on average than random hexamer primers. | ||
*#Use longer reverse transcription reaction times. | *#Use longer reverse transcription reaction times. |
Revision as of 13:24, 9 October 2007
See Q-PCR for much useful information.
This page focuses on information specific to quantitative reverse transcriptase PCR rather than quantitative PCR in general.
Protocols
- qRT-PCR/Two tube (in progress)
- qRT-PCR/Single tube
Notes
- The most commonly used specialist reverse transcriptase enzyme for cDNA production is AMV reverse transcriptase. It has RNase H activity (so that RNA molecules are only transcribed once) and has a high temperature stability (to reduce RNA secondary structure and nonspecific primer annealing).
- Since RNA can degrade with repeated freeze-thaw steps, experimental variability is often seen during successive reverse transcription reactions.
- Reverse transcriptase enzymes are notorious for their thermal instability. Repeated removals from the freezer can degrade the efficiency of the enzyme.
- Producing total cDNA from total RNA can be advantageous because
- cDNA is more stable than RNA so making total cDNA allows you to make multiple sequence-specific RNA measurements.
- This approach could reduce experimental variability from RNA degradation.
- To make total cDNA
- Use a polyT primer (most but not all eukaryotic mRNA) or random decamers (prokaryotic mRNA).
- Random decamers give longer cDNAs on average than random hexamer primers.
- Use longer reverse transcription reaction times.
- Ensure that the concentration of deoxynucleotides doesn't run out.