reverse transcription from viral rna - (Feb/25/2007 )

I'm very new in this field so I hope everyone can help me out with this basic question. I want to synthesize cDNA from Newcastle Disease Virus (NDV) - single stranded negative sense RNA. So how do I go about designing the primer for reverse transciption PCR?

What's the purpose of this project? Gene specific amplification?
Apart from that, just go about desiging your primer as you would for any RT-reaction, as it's an RNA virus, you might have to take into account diversity among different strains, so try to pick a region as conserved as possible.

yup.. i want to amplify the fusion and HN gene from NDV.. so i have to use oligo dT as primer to synthesize first strand and the reverse and forward primer that are specific to the genes right?

Hi,
I do RNA extraction and RT-PCR from Newcastle and Influenza virus.
I used the reverse primer (from the Forward and Reverse I use to amplify the gene) in the RT step. I think you can use oligo dT too, but if know the sequence of the gene of interest in the virus you have, may be using the reverse oligo would be better.

Actually, now I use an RT-PCR one step kit.
Buffer plus primers (mix of forward and reverse) plus RNA, place in the thermocycler and wait around 3 or 4 hours depending of the size of the genes.
I have better yields than using RT and PCR as separated steps.

Thanks!!

Using gene specific primers and one step RT-PCR as wel, works very good, higher yields and sensitivity than before.

I use:
SuperScript. III One-Step RT-PCR System from Invitrogen.

As I said, the buffer has MgCl2 and dNTPs already. In addition, superscript III has advantage over the “normal” superscript. You can check it in the datasheet.

It is easier, faster, less chance of contamination, and the yield is really much higher!

I think one-step kit from other brand should work well too, but this is the only I know.

Use the high fidelity version of the same kit.

I use:
SuperScript. III One-Step RT-PCR System from Invitrogen.

As I said, the buffer has MgCl2 and dNTPs already. In addition, superscript III has advantage over the “normal” superscript. You can check it in the datasheet.

It is easier, faster, less chance of contamination, and the yield is really much higher!

I think one-step kit from other brand should work well too, but this is the only I know.