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Real-Time RT-PCR one-step and two step issue - (Feb/26/2009 )

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Hi all, I'd like to express snoRNAs (about 90nt) in mammalian cells and want to detect its expression by RT-PCR. I'm not sure can ordinary RT-PCR work for this purpose. Should I use random hexamer or gene-specific primer? I also plan to do northern blot in the future. Do I need to run acrylamide/urea gel or ordinary denatured agrose gel?
Thanks so much!


In theory, amplicon in qPCR can be as small as 60-70bp, but question is if you can find suitable primers on such small area. I'm not sure about random hexamers, if they don't bind in your 90 nt at all, you won't get a product, so probably gene specific would be better. 2% agarose gel should have suitable resolution for 50 bp, but I'm not sure what bp difference it would be ale to distinguish.


I have used Qiagen one step PCR kit for PCR with DNA as template. Actually, the initial 95C step of conventional PCR will deactivate RT. In that case we can easily use one step RT PCR kit for amplification of DNA.


how can i extract RNA from blood quickly for RT PCR and RT LAMP ?

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