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1 year ago

Will presence of EDTA effect cDNA synthesis

I intend to use iScript cDNA Synthesis Kit in order to synthesize cDNA for qPCR. I have confirmed that my RNA is pure however, according to my extraction protocol I have suspended the RNA in TE buffer containing 1mM EDTA. Will the presence of EDTA have an effect on cDNA synthesis?

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There is a chance that EDTA might interfere with cDNA synthesis. In order to avoid that you could increase the volume of the cDNA reaction to 50 μl and add extra Magnesium. Make sure that you do not heat the sample in the presence of Magnesium though and only do it before adding it. If this does not work you should use the designated elution buffer of your kit after extracting the RNA instead of TE buffer. Alternatively, you can also use RNAse free water. Hope this helps.

Answered 1 year ago


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Hi, the EDTA acts as a metal chelator and will inhibit the activity of most enzymes, including reverse transcriptase. I would do a bead clean up and elute your RNA 10 mM TRIS HCL and 50 mM Nacl solution, or something to that end with a neutral pH. You can use the agencourt RNA XP beads for the bead cleanup by beckman coulter.

Answered 1 year ago


There is a chance that EDTA might interfere with cDNA synthesis. In order to avoid that you could increase the volume of the cDNA reaction to 50 μl and add extra Magnesium. Make sure that you do not heat the sample in the presence of Magnesium though and only do it before adding it. If this does not work you should use the designated elution buffer of your kit after extracting the RNA instead of TE buffer. Alternatively, you can also use RNAse free water. Hope this helps.

Answered 1 year ago


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