Real-time PCR vs. Northern Blotting / mRNA stability assays... - Advice from those who have experience with mRNA stability assays, plea (May/18/2007 )
Hi!
Just looking for everyone's opinion on using real-time PCR vs. Northern blotting. Is Northern blotting 100% "out" now for people to use?
I'm doing mRNA stability assays using Actinomycin D along with a drug to inhibit a kinase. I want to see if the level of the transcript goes down faster in the "+drug+ActinoD" compared to the "-drug+ActinoD". Does anyone have advice on which technique is best to use? I'm currently have a few results where I see the Cp values going to higher numbers (e.g., 25 --> 27) in the "+drug+ActinoD" samples, but whenever I normalize back to actin, a lot of the effects are gone 
I start with the same amount of RNA for the cDNA reactions, and treat the samples identically. Do you think I should normalize to various housekeeping genes just in case the actin is being affected as well in these treatments? Is it a total fluke that before correction to actin that my Cp values are *always* lower in the "+drug+ActinoD" samples for the target gene?
Opinions would be appreciated!
Thanks!
MapleCanPCR
Just looking for everyone's opinion on using real-time PCR vs. Northern blotting. Is Northern blotting 100% "out" now for people to use?
I'm doing mRNA stability assays using Actinomycin D along with a drug to inhibit a kinase. I want to see if the level of the transcript goes down faster in the "+drug+ActinoD" compared to the "-drug+ActinoD". Does anyone have advice on which technique is best to use? I'm currently have a few results where I see the Cp values going to higher numbers (e.g., 25 --> 27) in the "+drug+ActinoD" samples, but whenever I normalize back to actin, a lot of the effects are gone
I start with the same amount of RNA for the cDNA reactions, and treat the samples identically. Do you think I should normalize to various housekeeping genes just in case the actin is being affected as well in these treatments? Is it a total fluke that before correction to actin that my Cp values are *always* lower in the "+drug+ActinoD" samples for the target gene? Opinions would be appreciated!
Thanks!
MapleCanPCR
i tried real time pcr for measurement of mRNA expression (%). i have a theme to compare the data with Dot-blot analysis and also with a reporter assay for protein production. in comparison i got higher values in qRT-PCR compared with RNA Dot blot analysis. dot blot or northern is not the accurate method to get actual data, whereas qRT-PCR is more reliable i think.
we use 2 housekeeping gene ...actin and pgk
Hi! Thanks for the response -- this was for mRNA stability? Just out of interest -- what is "pgk" -- sorry, I'm still getting used to the acronyms on this forum 
thanks!!