I have carried out RNA extraction and my total yield is 1microgram which is in 20microlitre depc water. So how much of this in microliter should I take for cDNA synthesis
Where my RNA should be 1microgram
Look at the protocol for your cDNA synthesis kit. What is the greatest volume of RNA you can use as input into the kit? If you just have one sample, you could use that much. If you have multiple samples, you should decide the amount based on your lowest concentration sample, so that you can put the same amount of RNA into each reverse transcription.
Some cDNA synthesis kits allow you to use less than 1ug. If that is not the case, you can use an RNA concentration kit and concentrate your RNA to reduce the sample volume to that recommended for cDNA synthesis. However, I have had similar situations where I proceed with the maximum volume I can take and that has quite often worked for me. Please check your kit protocol first though.
I have been using cDNA kits from Applied biosystems (high fidelity) as well as Thermofischer (Verso). Both of them allows you to convert a minimum of 250ng of RNA into cDNA (I have tried and it works).
The main point you need to remember when you further use the cDNA for qPCR is that you need at least 50ng of cDNA.
So when I use 1ug of RNA for cDNA conversion, the total reaction volume is 20ul (including RNA + cDNA conversion components + nuclease free water). Once the conversion is done I add 80ul more NFW which gives me 1000ng of cDNA in 100ul. Meaning I have 50ng of cDNA in 5ul. Further for qPCR I use 10ul SYBR master mix, 0.4ul of each forward and reverse primers and remaining 4.2ul of NFW making the reaction volume to 20ul.
You can always reduce the reaction volumes if you have lesser RNA to start with.
Hope this helps.
Best!
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