Talk:Real-time PCR: Difference between revisions
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==Thanks== | ==Thanks== | ||
[[Image:Sunflower thanks.png]] Mille grazie a Mile. Vielen Dank an Sandra. Thanks for getting me started with Q-PCR. [[User:Jasu|Jasu]] 10:37, 26 March 2007 (EDT) | [[Image:Sunflower thanks.png]] Mille grazie a Mile. Vielen Dank an Sandra. Thanks for getting me started with Q-PCR. [[User:Jasu|Jasu]] 10:37, 26 March 2007 (EDT) | ||
==Reference genes== | |||
===Stability=== | |||
*'''[[User:Ajeffs|Ajeffs]] 06:55, 21 April 2007 (EDT):''' In addition to the given requirements of good (well, acceptable) specificity and efficiency of the reference gene primers, the next most important aspect of reference gene selection is stability. I don't care if the CT value of my reference genes (yes, genes, not gene) is close to the target genes/s or not - as long as the efficiency of all the primers is similar, and they are all working within their respective limits of detection i.e. linear range, then the stability of the reference genes between samples, treatments, etc. is the most crucial aspect of generating believable qPCR results. | |||
===Selection=== | |||
*'''[[User:Ajeffs|Ajeffs]] 06:55, 21 April 2007 (EDT):''' Screen a handful of ref genes, select the most stable using genorm, bestkeeper etc, use at least 2 reference genes for subsequent reactions and normalisation. Inlcude your genorm M values when publishing qPCR data. | |||
===18S=== | |||
*'''[[User:Ajeffs|Ajeffs]] 06:55, 21 April 2007 (EDT):''' 18S is generally a terrible choice for a reference gene thanks to the combination of (i) high abundance (creating a 1:100 dilution of template to run in parallel with neat template just for 18S is a complete drag); and (ii) having different degradation characteristics to mRNAs (it appears to be more resistant to degradation). However, if you can show that you have screened 5-10 reference genes, and 18S is still the best for your specific situation then so be it (but do try 28S if you or you PI is hung-up on 18S). | |||
Revision as of 03:55, 21 April 2007
The Babel effect
There are several names for this technique. I chose Q-PCR because:
- it's short (compare: quantitative reverse transcriptase PCR = QRT-PCR or RT-RT-PCR)
- it indicates what this technique is about: quantification (would you know what the technique is for if you had never heard about it and just read the term "real time PCR"?)
Q-PCR with previous reverse transcription could be put separately but since Q-PCR is usually preceeded by RT, I thought it best to keep things together here. Jasu 13:47, 26 March 2007 (EDT)
Thanks
Mille grazie a Mile. Vielen Dank an Sandra. Thanks for getting me started with Q-PCR. Jasu 10:37, 26 March 2007 (EDT)
Reference genes
Stability
- Ajeffs 06:55, 21 April 2007 (EDT): In addition to the given requirements of good (well, acceptable) specificity and efficiency of the reference gene primers, the next most important aspect of reference gene selection is stability. I don't care if the CT value of my reference genes (yes, genes, not gene) is close to the target genes/s or not - as long as the efficiency of all the primers is similar, and they are all working within their respective limits of detection i.e. linear range, then the stability of the reference genes between samples, treatments, etc. is the most crucial aspect of generating believable qPCR results.
Selection
- Ajeffs 06:55, 21 April 2007 (EDT): Screen a handful of ref genes, select the most stable using genorm, bestkeeper etc, use at least 2 reference genes for subsequent reactions and normalisation. Inlcude your genorm M values when publishing qPCR data.
18S
- Ajeffs 06:55, 21 April 2007 (EDT): 18S is generally a terrible choice for a reference gene thanks to the combination of (i) high abundance (creating a 1:100 dilution of template to run in parallel with neat template just for 18S is a complete drag); and (ii) having different degradation characteristics to mRNAs (it appears to be more resistant to degradation). However, if you can show that you have screened 5-10 reference genes, and 18S is still the best for your specific situation then so be it (but do try 28S if you or you PI is hung-up on 18S).
