Smaller than expected Bioanalyzer peak for size-selected RNAs?

[kerhard]

Hello all,

I just got some strange Bioanalyzer results from their small RNA chip, was wondering if anyone had experienced anything similar. My intention is to make small RNA libraries from these samples, so I wanted to get a good idea of the input concentrations.

The RNA samples were size-selected from an 18% denaturing polyacrylamide gel - I cut out the region between ~25-30 bp as far as my size marker indicated as can be seen in the attached image - but the results from the Bioanalyzer indicated that the vast majority of my samples are 20 bp in size. Huh?

Is this just an indication of degradation of my samples? I would think that if this were the case, there would be a lot of smaller fragments present in the Bioanalyzer trace as well.

Any ideas on this would be greatly appreciated.

Karl

[kwaraska]

I agree with your assesment that there would be more on the Bioanalyzer if it was degraded.

Bioanalyzer in our hands can shift sizes by a few up to 10 bp, but normally that isn't a shift you would see.

I wouldn't worry too much. It is still the population you want regardless of the exact size, right?

[kerhard]

I agree with your assesment that there would be more on the Bioanalyzer if it was degraded.

Bioanalyzer in our hands can shift sizes by a few up to 10 bp, but normally that isn't a shift you would see.

I wouldn't worry too much. It is still the population you want regardless of the exact size, right?

Unfortunately, size is the main determination of my population of interest. If I were to make a library from this material, it would likely be strongly biased for miRNAs, which is not what I am interested in.

A shift of 5 bp in the Bioanalyzer run would certainly explain this result. I guess it's a matter of whether I believe the Bioanalyzer results or whether I believe the resolution of the gel.