I made an extraction of total RNA with mirvana kit of life technologies for read miRNAs from cell samples, occur that the samples stayed with a low concentration (with 200 ng/uL or even less), but the integrity and relation 260/280 were good.
When I run the qPCR to evaluate miRNA gene expression it is ok, because I need a concentration of only from 10 to 20 ng per sample for construct my cDNA for this purpose. The problem is when I try to construct cDNA for run qPCR for gene expression of RNAm, because in the normal protocol we use generally 2 micrograms of total RNA in a volume of 8 microliters (sample+water), but in this case I only can use around 500 ng, otherwise the total RNA volume to construct the cDNA will be high.
When I run the qPCR with a cDNA construct with 500 ng of total RNA extract in this way (using miRvana kit), the housekeeping gene expression is ok (beta-actin, the CT is around 15), but the expression of my target gene simply doesn't appear (CT at 36 or undefined).
So my question is: what is the minimum amount of total RNA that can be used to construct cDNA by superscript II RT protocol that the target gene will express?
You can evaporate the water from the RNA-samples to increase the amount of RNA per volume.
Good luck ;-)
in our experiments usual for ds-DNA we choose the 5*10-5 M-1. but for FT-IR measurement we evaporated the water solvent of solution. however I am agree by Ninon as well.
The reason is populations of endogenous genes are mostly more than ur gene of interest only way to find optimal template for qpcr is to do by adding increased vol of template and see which can give u better expression
Thank you for your answers. Yeah Ninon, I thought in evaporate the water from my samples to concentrate it, but doing that I will lose some mRNA that I don't want to lose.
Well, thank you all for your sugestions. I discovered that the target gene that I want to quantifying in my case doesn't express that gene, so this is the cause that I don't see expression. But when I use 500 ng of RNA to quantify other knowed gene that is expressed in this kind of cell, I can see its expression. So, it is possible to quantify using only 500 ng of total RNA by this protocol.
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