Hi all,

I just did my first RP-SISPA. I extracted RNA from two virus species by different methods. One virus genome is ssRNA of 3 kb and the other virus genome is segments of dsRNA (3, 4 and 6 kb).

I used the two typical primers:

- TAG-6N

- TAG

Briefly the protocol is:

1) 4 ul RNA (most of the cases less than 10 ng) are reverse-transcribed by Superscript III (Invitrogen) with the primer TAG-6N as follows: 5 min 25C, 30 min 50C, and 5 min 85C.

2) the whole cDNA reaction volume is taken to perform single-trand synthesis with Klenow fragment (NEB) and added more dNTPs and more TAG-6N primer. Extension at 37C for 1 h. Termination at 75C for 10 min. No purification of this product was performed.

3) PCR of the ds-cDNA by using Dream Taq DNA pol (Fermentas) with 100 nM of the TAG primer. The program is as follows: 3 min 94C - 35 cycles of (1 min 94C - 1 min 65C - 2 min 72C) - 13 min 72C.

35 ul out of 50 ul of PCR product was analyzed in a 1% agarose gel.

My question is:

Is it expected that I'm getting more or less discrete bands around 1 kb?

Should I be getting a long smear with more intensity in the center?

Is this related with the fact that I'm working with two specific RNA virus isolates?

Thanks a lot!