Small amounts of RNA for qRT-PCR - (Jun/09/2006 )
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?
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.
no doubt that these assays can be expensive. Scale down strategies for RNA can really conserve precious RNA such as clinical samples (priceless!)
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.
Is there a generally accepted Ct range for qRT PCR for reduced error?
there are varying opinions, but I trust my data between 10 and 30
it is not the same for every amplicon, however. you need to determine the template input range that gives you the best efficiency within a reliable Ct range
i usually use 1µg of RNA for a cDNA conversion. depending on the gene, most of the time i get amplifications down to 1ng cDNA.
But even using RNA less and 200 ng still works fine, as long as your quality is high - thats it. Sometimes i also have amounts of less than 1 µg but i still can easily amplify my genes.
Just make a dilution series from your RNA/cDNA and see how far you can go down. Then you can adjust your amount according to your actual need.