Dear Scientists & Researchers,
I am going to amplify a specific gene from a (-)ssRNA virus.
I have collected several infected tissues and extracted their Total RNA with RNX-Plus solution.
Then I have used RT-2 Step cDNA synthesis Kit from Vivantis to synthesize cDNA with both first Random Hexamer and second Gene-Specific primer.
The length of my desired Gene is about 1500 BPs.
After cDNA synthesis step, I use 2 ul of produced cDNA as PCR template to amplify the Gene with Gene-Specific primer.
Nevertheless, no PCR product is shown on electrophoresis Gel, in exception a long pale smear in entire Gel columns (primer BLAST analysis shows no non-specific binding).
I think despite setting up the annealing temperature, the amount of Viral RNA in the preliminary sample is too low for detection with typical PCR.
How can I get sure about complete cDNA synthesis of Viral RNA Gene in the first step?, And also, how can I separate viral RNA from total RNA extracted?
Did you try to run a electrophoresis gel of the cDNA, to see if you have synthesized well the cDNA?
To increase the sensitivity of reactions, you should use RT with specific primers (by the way, did you used only one complimentary to (-)RNA primer? maybe it will be good idea if you use 2 primers - one to (-) strand, the second - to (+) strand of virus RNA - some viruses have a low quantity of their genome (-)RNAs in sample but a lot of (+)RNAs copies used in virus life circle) And, of course, nested PCR is more sensitive too.
Hi Roksaneh,
If you have a smear in your electrophoresis, that is indicative of presence of genomic DNA. Meaning you do not have pure RNA and hence cDNA. Meaning something has gone wrong with your RNA-extraction/purifiction procedure.
If I were you, I would do a different RNA-extraction method next to your RNX-Plus solution first.
Second, do you do the cDNA synthesis with both hexamer and gene-specific primer in a single tube? Have you performed these separately next to combined version? My suggestion would be to try this separately if you have not done this so far next to the combined version.
Good luck,
Ali
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