RNA RNA EMSA - (Dec/16/2016 )

Hello

I want details regarding RNA-RNA EMSA. I require your valuable suggestion for my problem with experiment.

I used two RNA for gel shift assay one of which is labelled with Digoxygenin. I get some shift in EMSA. I also perform the same thing with unlabelled RNA and perform EMSA assay as previous one and stained my gel with Ethidium Bromide but not able to detect any shift.

I am not able to conclude anything out of it.

Kindly suggest regarding

Waiting for reply

Regards,

Hasmatbanu Buchad

-hasmat buchad-

Your shift is relative to what? DIG gives a size shift relative to unlabelled as the DIG labelled nucleotide is bigger than a normal nucleotide. If you are comparing solely to a ladder, then the shift might be due to the DIG.

-bob1-

hello thanks for a reply

I expect sRNA-mRNA complex as shifted band of labelled sRNA.

-hasmat buchad-

It might be that your band is below the detection limits for ethidium bromide. You could try using a more sensitive RNA stain such as Sybr Green II or Sybr gold.

-bob1-

Hello

Thanks for a suggestion but it's a very expensive for my lab. Is there any other cheap alternative?

-hasmat buchad-

What about a probe based assay - labelled oligonucleotide that binds to the RNA, similar to a northern blot.

-bob1-

hello thanks for your reply .. I used labeled 1 RNA and incubated with unlabelled RNA to check RNA-RNA Interaction but no shift is observed...

-hasmat buchad-