Unexpected Bioanalyzer profile after Ribo-Zero bacterial rRNA removal

[bernardo_bello]

Hello,

Two doubts. I hope someone can give their opinion. I'm working with total RNA extracted from a sample that comes from a pig infected with our gram-negative bacteria. We are interested in RNA from bacteria. We have previously centrifugated in vivo sample to obtein only bacterial cells.
After this, we used Ribo-Zero gram negative bacteria kit to remove rRNA from total RNA extracted with phenol-clorophorm method.

1- I have a strong band after rRNA removal. See Bioanalyzer file attached. I would like to know if you recomend me to sequence this sample or do a fragment size selection (open to other alternatives). We are interested in total bacterial RNA and also in their sRNA.

2 - We still have not treated the RNA extracted sample with DNAse. When and how do you recomen to do it.


Thank you very much, Bernardo

[pmiguel]

(1)The "strong band" you circle is small RNA. Well, on the large end of small RNA, like tRNAs. Up to you whether you sequence it or not. But probably, you do not want to. If you heat/divalent cation fragment your RNA and then do a size selection (using AmPure, for example) you should be able to get rid of it, for the most part.

For miRNA sequencing, you would essentially do the opposite size selection. Removing any products with insert sizes above 40 bases or so.

(2)I like the "on-RNA-clean-up-column" DNAse digestion using Zymo kits. (Qiagen also has protocols for doing this.)

--
Phillip

[bernardo_bello]

Thank you for your response pmiguel,

(1) As I understand, there is no way to have a good sequencing coverage without doing a size selection. Thus, as you told me, there are two ways to know all sequences in this sample, normal RNA and miRNA.
I would like to have a detailled Materials an Methods of what I need to go towards those two approaches.

Tell me about heat/divalent cation fragment technique and the AmPure for the normal RNA sequencing.

Tell me what I need for miRNA selection.

I accept a paper as an example!

(2) Is the "on-RNA-clean-up-column" used before or after rRNA removal?


Thank you for your technical help.

[pmiguel]

Hi Bernardo,

I think you have the right idea about what topics to pursue. The information you seek is all available via web searches. You could start with illumina.com and their TruSeq RNA prep protocol and search the Zymo web site for protocols involving RNA clean-up.

--
Phillip

[bernardo_bello]

Thank you pmiguel, I will review those documents.

Another question,

What do you think about doing a fragment selection in gel after cDNA syntesis? It can be a good alternative to the options that oyu told me?

Bernardo

[pmiguel]

Sure, that is what you would do if, for example, you used the Illumina TruSeq small RNA kit to create a library.

For a TruSeq RNA prep, you would just use AmPure.

Another method:
With Zymo columns there is a procedure that can be used to wash away small RNA. You could use that, and attempt to re-capture the small RNA somehow.

--
Phillip