3 replies to this topic

[jas2311]

Hi
I need to detect whether my RNA of interest is present as dsRNA in vivo. I can detect both sense and antisense transcripts (more than 2kb) from the same samples using RT-PCR. I wanted to use a combination of RNases to detect the strandedness of these RNAs. Does anyone know whether Trizol is effective in maintaining the dsRNA during extraction? What about column based approaches like Mirvana?
Anyone has any experience working with DNA:RNA hybrid molecules?

Thanks in advance

[WSN]

If the RNA are in duplex format, you won't be able to get RT-PCR amp from them by doing RT at 50dC or even lower, unless you pre-denature them before RT.
sounds like you're doing in vitro detection, if it's real in vivo, you might want to use molecular beacon approach.
actually deep sequencing data revealed that most of the genome DNA are transcribed from both directions, so most RNA species have their reverse complimentary strands to go with.

[jas2311]

Thanks for the reply WSN. Before RT, I denature the RNA +primer mix at 65dC to avoid any secondary structure in RNA. Will 65dC denature the 2 RNA strands? Maybe.

[jas2311]

Could you elaborate a little more about the Molecular beacon approach to detect dsRNA?
Thanks