Small amounts of RNA for qRT-PCR-Real-Time PCR
I have a quick question. In our lab we have had a great deal of sucess using qRT RT-PCR on the lightcycler. We are currently trying to examine gene expression in exfoliated epithelial cells, however the cell numbers are such that we only get a very small amount of RNA <1 ug per sample. Typically we have been running our quantitative PCR using .1 ug of RNA per gene but we are considering going down to .01 or even .001 ug of RNA so that we can examine more genes with a single sample. Everything we run is normalized to a panel of 3 housekeeping genes.Has anyone had good experiences with using small amounts of RNA for quantitative PCR? If so, how low did you go? -cancergeek-
I think lower concentrations should work accurately. I've done quantitative assays RNA inputs for RT's as low as 15ng/sample. That was diluted 1:10 and used at a final concnetration of 0.27ng of RT in the QPCR rxn. I think it also depends on your detection equipment. The ABI 7900 is quite sensitive. I'm not familiar with the lightcycler.
-vasussci-
QUOTE(vasussci @ Jun 9 2006, 09:49 AM) [snapback]54846[/snapback] I think lower concentrations should work accurately. I've done quantitative assays RNA inputs for RT's as low as 15ng/sample. That was diluted 1:10 and used at a final concnetration of 0.27ng of RT in the QPCR rxn. I think it also depends on your detection equipment. The ABI 7900 is quite sensitive. I'm not familiar with the lightcycler. Not too hot on lightcycle but i agree with vasussci. the reactions can be scaled down. we used 50ng of cDNA per well and they worked very well in the ABI7700. we also scaled the volume of the reaction down from 50 to 12.5 ul whcih also saves a lot of money on reagents. -JPStewart-
no doubt that these assays can be expensive. Scale down strategies for RNA can really conserve precious RNA such as clinical samples (priceless!)
-vasussci-
I use 10 ng cDNA per qPCR reaction with good results. Have you tried making serial dilutions of your cDNA to determine your quantiative range? I have found that I can get acceptable Ct values down to .01 ng cDNA.
-soluene-
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