Hello, I am proposing to use quantitative reverse transcriptase PCR to analyze levels of a particular protein in human culture cells. I know this technique is faster then a northern blot and can be used on low abundance messages but what are the potential drawbacks of this technique? How much can you correlate mRNA level to protein level? For that matter, does this technique tell you the number of transcripts you had to start or does it just give you a value normalized to some house keeping gene?
Thank you
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#2
Trof
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Posted 17 May 2010 - 07:15 AM
qPCR is now a standard method to measure the amount of transcript, and AFAIK it's more precise than Northern blott.
Usualy mRNA levels are correlated to the amount of protein pretty well, unless there is some translation regulation involved.
You can do two basic types of quantification of mRNA. First, called absolute quantification, can tell you the number of transcripts present, but requires set of standards with known copies of the target gene. These are usualy harder to get.
Second, relative quantification only compares your samples to a control sample, and can tell you if there is increase or decrease in mRNA level between them (like, between untreated and treated cells).
Usualy mRNA levels are correlated to the amount of protein pretty well, unless there is some translation regulation involved.
You can do two basic types of quantification of mRNA. First, called absolute quantification, can tell you the number of transcripts present, but requires set of standards with known copies of the target gene. These are usualy harder to get.
Second, relative quantification only compares your samples to a control sample, and can tell you if there is increase or decrease in mRNA level between them (like, between untreated and treated cells).
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mcb56
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Posted 17 May 2010 - 11:18 AM
Thank you. That was helpful.
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sssbio
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Posted 29 May 2010 - 02:10 PM
Trof, on May 17 2010, 04:15 PM, said:
qPCR is now a standard method to measure the amount of transcript, and AFAIK it's more precise than Northern blott.
Usualy mRNA levels are correlated to the amount of protein pretty well, unless there is some translation regulation involved.
You can do two basic types of quantification of mRNA. First, called absolute quantification, can tell you the number of transcripts present, but requires set of standards with known copies of the target gene. These are usualy harder to get.
Second, relative quantification only compares your samples to a control sample, and can tell you if there is increase or decrease in mRNA level between them (like, between untreated and treated cells).
Usualy mRNA levels are correlated to the amount of protein pretty well, unless there is some translation regulation involved.
You can do two basic types of quantification of mRNA. First, called absolute quantification, can tell you the number of transcripts present, but requires set of standards with known copies of the target gene. These are usualy harder to get.
Second, relative quantification only compares your samples to a control sample, and can tell you if there is increase or decrease in mRNA level between them (like, between untreated and treated cells).
Hi,
I need your help regarding quantification of mRNA expression levels of trageted genes in controll and treated samples. I have no choice of reference housekeeping genes for relative quantification as these genes are showing significant down regulation in treated samples. So what are other methods that I can use to quantify mRNA levels?
I will be very happy if you guide me for this.
Thanks
