Real-time PCR for mRNA stability - (Jul/22/2008 )
Hi,
I am looking at mRNA stability (for my gene of interest) as a part of my work. My question is, can we use real-time PCR (SYBR Green-based) to assess mRNA stability under various conditions? The real-time PCR determines (or reflects) the abundance of the message of interest in a sample. But, some colleagues are not convinced about this rationale. They insist that northern blot or copy number PCR should be done to assess mRNA half-life. Any one who have used real-time PCR to determine mRNA half-life please share your thoughts here. Opponents of the approach, please share your thoughts too.
-jude0009-
QUOTE (jude0009 @ Jul 22 2008, 12:20 PM)
Hi,
I am looking at mRNA stability (for my gene of interest) as a part of my work. My question is, can we use real-time PCR (SYBR Green-based) to assess mRNA stability under various conditions? The real-time PCR determines (or reflects) the abundance of the message of interest in a sample. But, some colleagues are not convinced about this rationale. They insist that northern blot or copy number PCR should be done to assess mRNA half-life. Any one who have used real-time PCR to determine mRNA half-life please share your thoughts here. Opponents of the approach, please share your thoughts too.
I am looking at mRNA stability (for my gene of interest) as a part of my work. My question is, can we use real-time PCR (SYBR Green-based) to assess mRNA stability under various conditions? The real-time PCR determines (or reflects) the abundance of the message of interest in a sample. But, some colleagues are not convinced about this rationale. They insist that northern blot or copy number PCR should be done to assess mRNA half-life. Any one who have used real-time PCR to determine mRNA half-life please share your thoughts here. Opponents of the approach, please share your thoughts too.
I would go with the northern blot. There are always many variables to consider when doing RT-PCR, especially real time PCR and I think that this would confuse the issue of whether your stability is caused by normal degradation of your messages or degradation of your cDNA, PCR not working etc. A northern blot is more direct, and i think it would be easier to interpret.
-smu2-
QUOTE (jude0009 @ Jul 22 2008, 09:20 PM)
Hi,
I am looking at mRNA stability (for my gene of interest) as a part of my work. My question is, can we use real-time PCR (SYBR Green-based) to assess mRNA stability under various conditions? The real-time PCR determines (or reflects) the abundance of the message of interest in a sample. But, some colleagues are not convinced about this rationale. They insist that northern blot or copy number PCR should be done to assess mRNA half-life. Any one who have used real-time PCR to determine mRNA half-life please share your thoughts here. Opponents of the approach, please share your thoughts too.
I am looking at mRNA stability (for my gene of interest) as a part of my work. My question is, can we use real-time PCR (SYBR Green-based) to assess mRNA stability under various conditions? The real-time PCR determines (or reflects) the abundance of the message of interest in a sample. But, some colleagues are not convinced about this rationale. They insist that northern blot or copy number PCR should be done to assess mRNA half-life. Any one who have used real-time PCR to determine mRNA half-life please share your thoughts here. Opponents of the approach, please share your thoughts too.
Hm, I cannot see how could copy number PCR outperform real-time?
-Pallas-