I have good 260/280 and 260/230 values for all of my RNA extractions for left ventricle guinea pig heart tissue. My concentrations are pretty low though. Range from 20ng/uL to 45ng/uL of RNA.
My end goal is RT-qPCR. I will be making cDNA first. If I add 200ng of RNA to the cDNA (High-capacity cDNA Reverse Transcript Kit from ThermoFisher), would the concentration of the cDNA now be half of what the RNA was?
Example, 200ng RNA input (which would be calculated to achieve 10uL volume input) per sample is put into a 20uL cDNA reaction. The final volume of the cDNA reaction is 20uL. 10uL of that is the 200ng of RNA and 10uL of that is the kit components (10xRT buffer, 25X dNTP Mix, MultiScribe Reverse Transcriptase, etc).
This means my 200ng/10uL RNA input comes out as 200ng/20uL cDNA output. Diluting my ng/uL by 2.
Is this correct?
Hi Shanea Estes
Yes, this is correct.
For samples that have an RNA concentration of 20 ug/ul, Using 10 ul for cDNA reaction (Total volume of 20 ul) will end with a final concentration of 10 ug/ul (Assuming 1:1 reverse transcription).
Best..
Hello Shanea Estes
How are you doing today? Hum, thinking about the RNA concentration, your extraction is yielding small quantities of RNA. Is that correct or did you expect more RNA quantity?
Your calculations are correct, despite the reverse transcriptase kit you are using is able to synthesize up to 2ug of RNA.
I believe if you improve the extraction, you can get more cDNA for you qRT-PCR assay. Most reverse transcriptase kits work with 1ug of purified RNA and the final concentration of the reaction varies from 50ng/ul to 100ng/ul of cDNA depending on the kit.
cDNA is too expensive to synthesize using only 200ng of RNA, and we usually work with diluted cDNA for each sample in replicates. Probably if your run a assay using this cDNA you will need to setup more than 35 cycles in the thermocycler to achieve the exponential phase of the reaction.
I recommend you improve your extraction to get more RNA from your samples. Try to use at least 1ug of RNA for your reverse transcriptase reactions. This will help you in you in the qRT-PCR analysis.
My best regards and good luck. ^^
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