ribosome depletion causing ribosome degradation?

[valid_name]

basic question concerning use of gram-negative Ribozero ribosome-depletion kit:

has anyone noticed this system create what appears to be significant degradation of ribosomes?

(input total RNA was in TE, following exactly provided protocol)

from attached bioanalyzer trace:
blue trace: RNA pre-Ribozero treatment, RIN = 9 23s/16s =1.6
red trace: apparent degraded ribosomes?

[pmiguel]

basic question concerning use of gram-negative Ribozero ribosome-depletion kit:

has anyone noticed this system create what appears to be significant degradation of ribosomes?

(input total RNA was in TE, following exactly provided protocol)

from attached bioanalyzer trace:
blue trace: RNA pre-Ribozero treatment, RIN = 9 23s/16s =1.6
red trace: apparent degraded ribosomes?

First, your plots are not correctly aligned (the spike-in control peaks do not overlap.)

Second, yeah I am not so sure that hill is ribosomal. Not what I would expect for degraded rRNA. Could be though.

--
Phillip

[valid_name]

First, your plots are not correctly aligned (the spike-in control peaks do not overlap.)

Second, yeah I am not so sure that hill is ribosomal. Not what I would expect for degraded rRNA. Could be though.

--
Phillip

ok... new user of bioanalyzer RNA! let's see if this helps any. with the peaks aligned, it looks like substantial 23s degradation; also spike at 200nt?

thanks for bearing with such elementary questions!

[pmiguel]

ok... new user of bioanalyzer RNA! let's see if this helps any. with the peaks aligned, it looks like substantial 23s degradation; also spike at 200nt?

thanks for bearing with such elementary questions!

Are you sure that you did not concentrate the depleted sample?

If 23S degrades you expect degradation products smaller than the original peak. Instead we see a hill on both sides of where the 23S was. Maybe that is the mRNA?

--
Phillip

[scooter]

Not degraded after Ribo-Zero

Actually, your RNA post-RZ looks exactly like it should. The higher molecular weight hump is like your mRNA and the lower set of peaks is likely to be abundant smaller RNAs like snoRNAs, tRNA, etc.....

If you call up Epicentre tech support they will likely tell you that they get calls with this issue all the time. Ribo-zero is so effective at removing rRNA that many people assume the worst at first glance. mRNA is only ~1-2% of a total RNA sample, so if you are removing ~99% of the rRNA you would not expect to see much on a BA trace after removal.