• The slow growth of your cells will not affect the quality of your resulting RNA; Just yield
• Regarding the ribosomal peaks, yes the maxim is that the 23S and 16S peaks are generally in a 2:1 ratio but that can vary
• RNA that is extracted after storage tends to exhibit the phenomenon of relative enrichment of the lower ribosomal sub unit compared to the 23S sub unit compared to RNA extracted immediately from fresh material
• Nevertheless, I would not worry about it too much: Not with standing the fact that it might be a reflection of your starting gram positive cultures, your peaks are sharp and not jagged and the signal to noise is high, i.e. you do not have peaks between the ribosomal subunits: This is why your5 RNA integrity number (RIN) is high (>8.0)
• I think in that regard your RNA is of high quality
• What doers however concern me is the peak muchyfurther down: This is prominent and might represent guanidinium salts that are an integral part of your lysis and protein denaturation buffer
• What is your 260/230 ratio ? if Less than 1.0 I would be confindent that what you have is a sample of RNA that is undegraded but unduly contaminated with GITC that could denature down stream reactions like RT synthesis
• If your 260/230 ratio is < 1.0, in concert with the low Mr peak I would try to remove the GITC salt (peak) by precipitating your RNA with 1/20 volume 3M NaAc pH 4-5 and 3 volumes of Ethanol @ -80C for > 1 hour
• Then spin, remove supernatant and vortex pellet with room temp; Repeat thei s70% wash before air drying pellet and resuspending in water at 4oC for >/=1 hour: That will remove salt and hence eradicate your low mr peak
• Degradation might not be the actual issue here !

@ Laurence Stuart Dawkins-Hall First of all, thank you for insights.

• RNA was extracted from samples collected ~2 weeks ago. The protocol says that homogenate can be stored at -20 or -70 C for at least two years. Compare to this my samples were stored relatively short period of time. Could it still affect RNA?
• 260/280 and 260/230 ratios are fine, ~2 and ~2,2, respectively.
• Moreover, I didn't mention what I have observed when analysed more RNA quality data: the lower 23S rRNA peak - the higher peak between 25 and 200 nt in length. It seems 23S rRNA is cleaved into small pieces. (see figure)
• I'm concerned that mRNAs can be lost in samples like 23S rRNA. Could it be true?

Dear Ramona

Yes it can but in my experience extraction from stored RNA will generally result in that lower 23s peak

To answer your question: no you will absolutely lose mRNA from your sample if the ratio of your rRNA changes; the 2 are completely independent

To reiterate your RNA - for stored RNA - is of high quality as your signal to noise is very good and your RNA is devoid if degraded products; hence your high RIN number

The additional prominent low Mr peak However does concern me: as somebody who has examined Agilent traces for 20 years and worked with RNA for 30 years in effect what I see when I look at your trace is relatively clean undegraded RNA but contaminated with GITC. That being so you do need to remove that peak (contamibation) by 're ppt your RNA as explained and then desalt with 70% ethanol x 2

Laurence Stuart Dawkins-Hall maybe from your experience you know something about extremely large peaks in 5S area? My total RNA doesn't look degraded, but somehow that peak is quite high.

Yes its GITC from lydia buffer and should be removed by re purification

Otherwise if that visible concentrations will be inhibitory to RT

Repurify your sample with a 2nd RNA column

When you do make sure you was with your ethanol based column was buffer x2 and before spinning through leave 1 minute at room temp_spin for 1 minute_apply more was buffer_leave 1 minute_spin 2 minutes then a further 1 minute to dry column. Apply millie water. Wait 5 min at room temp. Spin to elite repurified RNA. This should reduce that peak

Dear Laurence Stuart Dawkins-Hall ,

Thank you for your reply. However, I am not particularly sure if GITC is the problem. As you can see in the picture 1 (I am very sorry that I didn’t add this earlier) the peak of 5S RNA and tRNA are quite high, even higher than 16S and 23S. However the ratio 260/230 is quite good – 2,17 (260/280 ratio is also okay as it is 2,05). I believe that if my samples were contaminated with guanidine thiocyanate (that is in RNAzol® RT – used for total RNA extraction) the ratio would be much higher as guanidine thiocyanate absorb at ~260 nm.

As the rRNA Ratio [23s / 16s]: is quite good (1,9 according to agilent, also it looks not bad in agarose gel – picture 2 – 1,6) we believe that we have a lot of 5S rRNA. What is more I got similar results from different 24 total RNA samples.  Approximately bacteria biomase was in the -80C freezer for 8 days. After that bacteria was physically lysed with liquid azote and mixed with RNAzol. Prepared samples were left in the -20 freezer for a month and then total RNA was extracted. Previously you mentioned that “RNA that is extracted after storage tends to exhibit the phenomenon of relative enrichment of the lower ribosomal sub unit compared to the 23S sub unit…..” . Could this be the problem? What do you think? Also, can I use these samples in later experiments?

Also, all samples are collected from bacteria late exponential/stationary phase. I read that in stationary phase E. coli contains large fraction of 5-monophosphorylated mRNAs that represents a particular state in the mRNA decay. Maybe that got something to do why I am seeing this enormous peak?

Thank you for you time and consideration