RNA quality problem - (Aug/31/2005 )

Please see the picture of RNA isolated from campylobacter.

I'm not used to RNA. but I think the quality is very low.
RT-PCR didn't show anything with this RNA.
This RNA is isolated by Trizol.

Please help me improve this.

Thank you in advance.

RNA quality might be okay.

I think you loaded way too much on the gel. How many micrograms/ lane did you load.
Bad RNA runs as a big blob at the bottom of the gel.
Did you run a 260/280 on them?

Did you include a positive control in the pcr? One you made from good RNA and from the same type of sample?

Thank you for your reply.

The quantity of RNA loaded on the gel is

left: 1ug
2nd to left: 2ug

and I think the rest two were degraded.

Do you think DNA is contaminated in the RNA sample?

Hello,
I havent worked with bacterial RNA. Human RNAs presents ribosomals bands so you can evaluate its integrity comparing the ratio of those bands. I dont know what pattern I should expect in your sample. Anyway I dont think your RNA is ok because if all the lanes contains RNA I can see that there is a different pattern in every one of them, so at least one of them is degradated.

Hi,

I've just started working with RNA. I recommend to go the Ambion website. They have whole bunch of information about RNA. Besides that, I think your RNA might be degraded. You should be able to see a band at about 3 kb (23S rRNA) and at 1.5 kb (16S rRNA). The intensity of the 23S rRNA should be about twice as much as the intensity of the 16S rRNA band. Also, check the A260/A280 ratio.

Good luck,
Susan