How much cDNA is made in a cdna reaction

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How much cDNA is made in a cdna reaction

Postby marcpaulo » Mar 15 2017 8:43 am

If you start off with 20ul of 20ng/ul of total RNA, and you need 4ul of this for a 20ul cDNA reaction (4ul template RNA + 16ul of enzyme and buffer mix).

My understanding is I've input 4ul of 20ng/ul total RNAthat's 80ng, so is the output 80ng of cDNA?
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Re: How much cDNA is made in a cdna reaction

Postby newbie17425 » May 25 2017 7:48 am

marcpaulo wrote:If you start off with 20ul of 20ng/ul of total RNA, and you need 4ul of this for a 20ul cDNA reaction (4ul template RNA + 16ul of enzyme and buffer mix).

My understanding is I've input 4ul of 20ng/ul total RNAthat's 80ng, so is the output 80ng of cDNA?


Which cDNA synthesis kit are you using?

I usually make 1000 ng of cDNA. For this, I divide 1000/RNA value obtained by nanodrop in ng/µl.

For example: For sample X, I obtained 500.5 ng/µl. Divide 1000/500.5 = 1.99 ~ 2 µl.

I'm using first strand cDNA synthesis kit from Thermo. So according to the protocol, I add 4µl of reaction mix + 2µl of enzyme mix + 2 µl of RNA + 12 µl of water for a final volume of 20 µl.

Hope this helps.
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Re: How much cDNA is made in a cdna reaction

Postby 29yrsExperience » Sep 08 2017 5:18 pm

You are starting with total RNA, so your cDNA output will depend on what primer (oligo-dT, random hexamers, or gene-specific) you use to do first strand synthesis. Oligo-dT will specifically prime mRNA with poly-A tails (a lesser component of your total RNA, which contains a lot of ribosomal RNA), while the random hexamers (or random whatever-length, it varies) can potentially prime anywhere, so the output is higher. Obviously the gene-specific primers should only reverse-transcribe the specific message, so the output in that case would be a tiny fraction of the starting RNA amount. Some “universal” RT kits have a blend of oligo-dT and random primers for maximum output. For random hexamers we usually “assume” the cDNA output is equal to the RNA input, but calculating the exact quantity you will get is not really possible. Just be consistent in how much RNA you add to all your reactions for a given experiment.
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