For cDNA synthesis, high-quality RNA with an A260/A280 ratio of 1.8 to 2.2 is ideal, and a minimum concentration of 1 μg/µL is usually recommended. For Real-Time PCR, the optimal RNA concentration is highly dependent on the gene of interest, and can range from 1 ng/µL to 500 ng/µL.

Yes you can, the kit is efficient to convert rna in to approx 1ug/ul concentration of cDNA

Dear Mit Joshi

First, the most important ratio regarding RNA is 260/230, and as this ratio represents both the concentration of RNA and the purity (salts...) of RNA, we have to consider the both factors. Actually, you have too low ratio. Ratios with lower than 1 may cause problems during reverse transcription. so it is better to precipitate your sample before taking further steps.

Regarding the concentration desired for conversion, most kits suggest not more than 1 ug of RNA, and practically it is better to reverse 800 ng if the highest limit of your kit is 1 ug. It is worthy to mention that reversing higher amount is better since it is more affordable and will give you a hand while performing RT-qPCR.

Hello Mit Joshi

The assessment of RNA integrity is a critical first step in obtaining meaningful gene expression data. Working with low-quality RNA may strongly compromise the experimental results of downstream applications.

So, you should check RNA integrity before gene expression. In addition to good quality template RNA (high purity A260/A280 > 1.8 and A260/A230 > 2.0), you should assess the integrity of total RNA by running an aliquot of RNA sample on a denaturing agarose gel stained with ethidium bromide. The intact total RNA run on a denaturing gel will have sharp, clear 28S and 18S rRNA bands for eukaryotic samples. The 28S rRNA band should be approximately twice as intense as the 18S rRNA band. This 2:1 ratio (28S:18S) is a good indication that the RNA is completely intact.

If I were you, I would not be happy with the RNA quality and would not go ahead with cDNA synthesis. I would get the RNA right and then proceed.

The concentration of RNA required to synthesize cDNA will depend on the expression level of the genes under study. There is always a range you can use which is usually within 100-1000 ng.

Best.