I assume you measured your RNA and reverse transcribed the same amount of RNA per condition e.g 1ug, so no you don't need measure your concentration again.

For qPCR, you want each sample to roughly have 50-100ng because its still an amplification process.

So if you have RT'd 1ug in 25ul. 1ul of your RT reaction should roughly be 50ng per qPCR tube.

As noted, you should assume 1:1 conversion. You can't easily measure it, because your cDNA prep will contain unincorporated nucleotides, leftover RNA, all sorts of other stuff that will render spectroscopy uninformative.

I typically find that 8ng per well (assuming 1:1 conversion) is sufficient even for qPCR of relatively low expression targets, and you should always start lower rather than higher: using too much cDNA can inhibit the PCR efficiency, and also wastes a lot more cDNA.

As Matthew de Cruz explained, that no need to measure again. You only need to measured RNA conc. and synthesize respective cDNA.

Also conc. of cDNA suggested is good enough.

Let's take you synthesized 1ug cDNA, then u can dilute the cDNA to 1:20 or 1:10 or 1:5 ratio dilution and check for endogenous housekeeping gene expression and standardized the dilution as well as conc. of cDNA for your PCR reaction. dilution is necessary for efficient PCR reaction as well as to avoid wastage as John Hildyard suggested.

Dear

I think you should assume the conversion of 1:1 from the total RNA especially in quantitative real time polymerase chain reaction. Regard

I don't think you can assume a 1:1 conversion rate or accurately measure the neat concentrations by spectrometry. If you are comparing two or more samples, it is reasonable to assume that, if you put the same concentrations of RNA into the reverse transcription reactions, then they will have close to the same concentrations of cDNA.

If you need absolute cDNA amounts, I'd add standards to the qPCR - e.g. 5X 10-fold serial dilutions of appropriate genomic DNA of a known concentration. The standards will give you a range of amplification curves from the qPCR. By comparing the standards against the sample, you can make an estimate of the starting concentration of your samples.

Thank you so much for your help

Saurabh Mandal why to dilute cDNA can you please clarify it thank you so much

Dear Basma Ali

Mostly neat cDNA will becomes very high in conc. for sensitive technique like qPCR and the use of diluted cDNA usually decreases the background noise, primer dimers. Even dilutions reduces the effect of inhibitors.

Also gene copy number, say high or low copy number of gene need to be considered, say high copy number gene can be diluted while for low copy number it is not necessary for dilution, so that Ct value should properly fall between 15 cycles and 25 cycles. Overall you have to decided the optimal dilution.

Thank you so much for your time and consideration