Is it possible to obtain a positive PCR result from RNA fragment without making cDNA?

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How do I increase the yield of my PCR product?

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How optimize the Size Exclusion Chromatography?

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Does anyone know HRM channel in a 2 plex QIAGEN Rotor- Gene Q qPCR can be used as a separate channel?

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How to address this issue in the analysis of Real-Time PCR results?

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Does anyone have the experience of using Taq Man probes in the QIAGEN Rotar- Gene qPCR machine?

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Expression cloning by using cDNA,do I need to modify the cDNA first?

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MiRNA qpcr problem?

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Runs of nucleotides in Sanger sequencing?

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Calculating volume of DNA required from DNA concentration?

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Mark B Stead

It is not possible for a standard DNA polymerase to reverse transcribe RNA, so you must have DNA contamination. I would suggest performing an acid phenol/chloroform/isoamyl alcohol (Ambion is a good source for this) extraction of the RNA sample, followed by a sodium acetate/ethanol precipitation. Any further DNA contamination can be removed by using the DNA-free kit from Ambion.

Standard RNA extraction methods are not very good at removing all traces of DNA, but the combination of acid phenol and DNase I works very well, but do the steps in that order.

Good luck!

Navonil De Sarkar

You may also go for DNase treatment followed by Purelink in-colum (Life tech) digested DNA removal. Its a very efficient method

Ali Farhoudi

Thanks, I am going to purify My RNA after 1st Dnase treatment , using acidic Pnenol/ cloroform/IAA purification and performing PCR on the eluted one and if again is positive I will perform second Dnase treatment on the column.