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A question for Reverse transcription experts


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5 replies to this topic

#1 Mohamed samir

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Posted 25 April 2013 - 02:13 PM

Hallo all,

I Have RNA isolated from murine macrophage. I want to creat cDNA. I have a kits from invitrogen that include no primers and no obvoius polyA tail polymerase Enzyme.

. The question is

Can the reverse transcriptase E act as ploy A polymerase at the same time under the same temperature.
Can I use the miRNA primers to creat cDNA for the miRNAs only but not all RNA ? is it critical to have an random primers or what



If I do this cDNA synthesis and I want to be sure that my product (total RNA ) get changed to cDNA, what is the accurate method for confirming this ? Can I measure the cDNA concentration after RT or this is not indicative ?

#2 bob1

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Posted 25 April 2013 - 04:11 PM

Usually these kits contain either an Oligo dT solution or a random hexamer solution that acts much like a single primer in PCR for the reverse transcriptase to bind to and then replicate the DNA. This process is pretty efficient, but I would say that it is never 100%. However, we have no real way of easily assessing the conversion other than to assay for known low abundance genes, but even that is problematic in that the systems used all rely on conversion to cDNA, so it is a circular argument.

Measuring the DNA content after transcription doesn't work as the dNTPs and any residual RNA will also give a reading on a spec.

#3 pcrman

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Posted 25 April 2013 - 09:02 PM

If you want to detect miRNA, you may need to use a different kit to isolate small RNAs because some column based kits are not good for isolating miRNA. miRNA RT requires special primers.

#4 Mohamed samir

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Posted 26 April 2013 - 05:10 AM

I have already primers for my target miRNAs but not Oligo DT primer. I mean is this is right to do RT for my target only using the miRNA primer assay so that I end up with cDNA for my miRNA but not all the RNA molecule. How can I be sure that the miRNA RNA became cDNA ?

#5 pcrman

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Posted 26 April 2013 - 07:34 AM

The question now is how you isolated your RNA. If the method of isolation loses miRNA, then you won't get miRNA converted to cDNA by miRNA RT reaction.

#6 Mohamed samir

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Posted 26 April 2013 - 01:57 PM

My dear it is not logic that Iam asking for cDNA synthesis without isolating miRNA sample




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