Not sure why it would be necessary since you have a very pure RNA sample and, I'm assuming of course, you're probably going to do some sort of transcriptomics (Reverse Transcriptase PCR, RNA seq, etc.) on that sample.  If you are going to do RNAseq on total RNA then bacterial RNA will present a problem, however if you're doing RNA capture, you'll be hybridizing with specific eukaryotic sequences, same with RTPCR in a sense.  I think the main question would be what is you downstream application?  As for determining if you have bacterial contamination in the sample you could simply do RTPCR using a bacterial primer.  The best bet would be to use a primer for the 16S ribosomal subunit, it will be present at a much higher concentration than other bacterial RNA's, therefore if that's not there than you have no need to worry about bacterial contamination.  Not familiar with the Agilent Tape Station, but I've used the Agilent Bioanalyzer 2100, and your RIN number is great, what were your concentrations like?  Anyway, hope this helps, and best of luck.

Hi Dave, thank you very much for your suggestions.

I will indeed sequence RNA and I had some serious troubles with bacterial contamination in my first samples that were sequenced. I got the answer from the Agilent machine responsible yesterday too. Apparently the Tapestation can not distinguish between bacterial and eukaryotic origin of the sample, it rather just shows the peaks of the detected RNA in the sample. And it's true that the RIN value is good compared to my previous samples where I had something between 2.5 and 5.0. I probably will do some qPCR with 16S ribosomal subunit to check for contamination. So thank you very much for your advice!

Hi Adisa, you're welcome.  Yeah, anything below RIN 7 generally doesn't produce good result.  A cheaper way to test would be using the nanodrop, but with low concentrations you're probably better off with using Agilent.  How did you do your extraction? Trizol, or column purification?  Also I was wondering where the contamination arose from.  We usually put samples in RNAlater immediately after dissection then snapfroze them.  Bacterial RNAses can degrade your samples a lot.

Hi Dave, thank you for your further comment.

Well, my first samples went through a rather complicated process: Perfusion of mouse, dissection of cerebellum, homogenization, FACS sorting of cerebellar cell homogenate, RNA extraction with bioline kit and then finally the RNA seq. Although the process was long, still I am wondering which step was the critical one for the bacterial contamination since around 70% of the identified RNA was of bacterial origin (Micrococcus antarticus).

With the new samples that will be sequenced the protocol is almost the same: perfusion, homogenization, cell sorting and rna extraction with Bioline kit. I had RIN values from 8.8 to 9.2 but only 300-530ng total RNA.

In general we don't use any of this RNA extraction additives, but we do pelett the cells and then resuspend and freeze directly into the lysis buffer of the extraction kit. I have the feeling that this improved the qualty as well since I didn't do that with my first samples that were also badly degraded beside the contamination. But still the sequencing worked out.

Dear Adisa,

Glad the sequencing worked out for you.  If I had to venture a guess, I'd say that the contamination arose during dissection and preparation of samples.    Provided all other processes were performed under a laminar flow hood with sterile technique, then further contamination shouldn't have been an issue.  Either way, it sounds like you have it under control now, good job, and all the best! 

Micrococcus antarticus, really?  LOL, I guess I know where your mice came from then:  https://www.ncbi.nlm.nih.gov/pubmed/10758880

Must've been stowaways at Concordia or McMurdo Station.

Storage in lysis buffer is a good solution.  300-530ng total RNA per how many uL?  Or out of how much initial sample?  Just out of curiosity, what method did you use to quantify the 70% prokaryotic contamination?