I would stay away from using the multi-beads shocker for extractions as they can shear the RNA. They are great for protein work and hard to digest tissues, but unnecessary and in my experience are potentially damaging for RNA/DNA extractions. I would second a lot of the other general comments by the other people who replied, that you should first stick to the protocol, second that you should always centrifuge RNA extractions at 4 degrees and finally that you should have a RNase free bench area with RNase free pipettes, etc. to do all of your work.

Hi,

You should perform all your centrifugations at 4°C. If the problem persists, try to use a freshly opened kit, or a new brand. If your protocol improvements include your own solutions, re-prepare them and treat with DEPC, overnight at RT.

I do believe that before having doubts, you should try the protocol suggested by manufacturers, and see if results are different. Anyway, as Bertrand suggests, you should be absolutely sure not to have degrading hand-made solutions.

You should be sure that your starting material is of good quality. Most RNA degradation occurs through endogenous RNAs that become active when cells/bacteria are death (e.g. because of slow thawing, or inadequate storage).

Most commercial lysis buffers have RNA inhibitors and these work fine. In addition, most of the subsequent steps are in relatively pure conditions. Small RNase contamination will only have minor effects. We have left isolated RNA withoug much precaution at RT for more than 24 hours without significant RNA degradation, indicating that in most cases the bulk RNA degradation happens in the sample before the extractino procedure.

I think you should reconsider what you do with your samples before the extraction. Why do you use RNalater? Direct freezing to -80°C is generally sufficient, easier and cheaper, but requiers quick thawing and direct lysis after storage.

Hi Cristina, I agree with a lot of what Bertrand says - 4 degrees is definitely important and restarting a kit etc. is always a good option in case you've got contamination. I personally don't use DEPC-treated water, but know that plenty of others do and it can be a useful approach.

Some other really basic tips that I always use when using RNA include: always wipe your bench down with water then etthanol before starting work; keep a separate set of pipette tips for use only in RNA experiments so you don't cross-contaminate; if you use your pipettes for non-RNA work it might be worth cleaning the barrels with RNAseOUT or just ethanol etc. in case you've got some RNAses in them; and always pour yourself a new aliquot of deionised/Milli-Q water at the start of each experiment rather than keeping one. I'm sure you do a lot of these already, but it's always worth mentioning just in case they spur an idea!

Good luck!

Another way is to try different isolation kit like Directzol from Zymo. It works for me really good.

Hi Cristina

My expertise is with plants. If you are concerned about mRNA degradation you could isolate the polysomes first. See ref 11 and 18 (and Davies and Abe Methods in Cell Biology). If you want to see the rRNA integrity at different stages of extraction, use GPS buffer (ref 11) and use electrophoresis. This will tell you if and when degradation occurs and help you decide what steps to modify. I would try to do all steps at 4C Best wishes, Eric

11. B.A. Larkins and E. Davies. 1975. Polyribosomes from peas V. An attempt to characterize the total free and membrane-bound polysomal population. Plant Physiol. 55: 749-756.

18. E. Davies and B.A. Larkins. 1980. 'Ribosomes'. In: Plant Biochemistry: A comprehensive treatise. Vol. I. pp. 413-435.

I would stay away from using the multi-beads shocker for extractions as they can shear the RNA. They are great for protein work and hard to digest tissues, but unnecessary and in my experience are potentially damaging for RNA/DNA extractions. I would second a lot of the other general comments by the other people who replied, that you should first stick to the protocol, second that you should always centrifuge RNA extractions at 4 degrees and finally that you should have a RNase free bench area with RNase free pipettes, etc. to do all of your work.

Follow the protocol outlined in this paper: PMID: 22130707 Amisten 2012, available on my RG page

Hi Cristina.

Your 23S rRNA is not neccessarily degraded as such.

Do you have a gel image that you can post here or send me via email?

This is a phenomenon you can see in some plant chloroplasts and I also see it in some cyanobacteria.

It is only the 23S that falls into two pieces at a very specific nucleotide sequence.

You can probably see two smaller fragmens around the 16S? These are the two products of the cut 23S.

If this is the case, you do not have to worry at all about you mRNA being degraded as well (unless you actually see a smear in your gel). :)

Best, Ulrike

If I were you I would resuspend the pellet in trizol and freeze at -80C before extraction. You could try extraction using liquid N grinding, never letting the sample defrost. I would agree with Michael that bead beating will shear your RNA, however whether that is a problem depends on what your experiment is about. We have not had good results using RNALater with our samples, but it should work for bacterial pellets.

I use a bead beater for extraction of some harder-to-crack cyanobacteria and the pattern of RNA on the gel is the same as without beat beating them. 23S falls into two pieces, nothing to worry about.

Still, I agree with Robert Forster about not using RNAlater. This is an option for field samples, but you do not need it in the lab. Put your samples directly into Trizol or PGTX (Pinto 2009:Analysis of current and alternative phenol based RNA extraction methodologies for cyanobacteria) and snap-freeze them before continuing with your work.

When using the bead beater for RNA, it should either have a good cooling device OR your samples should be in some phenolic solution to stop internal RNases from degrading your RNA.

Thank you for all the answers and advices.

I've recently joined a new group where former colleagues adapt the protocol for RNA extraction for RNA-seq analysis.

I've of course taking all technical precautions, changing gloves frequently, clean RNAse-free bench, RNAase-free water and reagents, in a separate and exclusive area. I even wear a mask (that I personally think is not really necessary, but...)

Anyways, I think that Ulrike might made the point to my problem.

I'm attaching here an agarose gel and the analysis using a Bioanalyzer of the correspondent sample.

Well, the protocol I used was optimized for former components of the lab, and until now seems to work well for the main purpose, RNA-seq, so at first maybe no change is necessary, but I will take into account all advices.

My supervisor thinks RNA marker is not really necessary because, in addition, there is no proper RNA marker on the market. This is surprising for me, but as I'm not yet familiar with RNA, what do you have to say about this?

uops...

There you are the bioanalyzer image.

Is this the same case as you have previously seen for cyanobacteria?

hi

I think for RNA extraction, time of extraction is important and you must use a kit that protocol of it must be done in minimum time.

using beads may be break long RNAs like 23srRNA.

Are you tried kits made by Qiagen company. in my experts RNA extraction by this company products would made proper results.

HI Cristina.

From your gel image I would think that you have very little overall degradation (see the faint smear below the 16S). But this could be due to the gel or your image...

Your electropherogram looks fine to me and there is no significant amount of degraded RNA. And yes, one of the two peaks in the size range of the 16S will be your 23S breaking products. I personally see two 23S fragments (see my gel image: top band 23S, followed by 3 bands around the 16S) and one 16S band, but you might well see just one, if it breaks into two pieces of similar size.

What I am more confused about in you bioanalyzer image is: where are your tRNAs (~60-80nt) and RNaseP (~350nt)? It looks like your kit omits smaller RNAs.

This is why I never use kits (but maybe you do not want or need the smaller ones).

This old timer never used kits to extract RNA, but in my experience degradation was correlated with the ratio of tissue, and likely the total protein within the sample, to the amount of chaotropic agents used in the prep. If you're using the same amount of buffer to suspend the tissue (cells), but different number of cells per sample, the ones with more cells will tend to be more degraded, because you're overwhelm the chaotropic agents, and a certain percentage of the RNAses remain active. The solution is to use fewer cells. Also, tons of phenol-chloroform solves most of these degradation problems; never been sure how anybody can manage RNA extractions in detergent alone.

Thank you Ulrike for your comments.

We are actually interested in mRNA, so tRNA and other smaller RNA are not interesting... My main concern was about degradation, because I was affraid that if 23S rRNA was degraded, mRNA might also be. But colleagues that are working in this project for a while told me that this phenomenon concerning 23 rRNA is consistent and characteristic of the bacterial strain we are working with.

In my project I'm also working with a second bacterial strain, so I will be able to confirm if the phenomenon is also happening in both of them.

Thank you again for your help, I'm a relieved now to learn similar situation occurs with other microorganisms.

By the way, can you recommend me a good RNA marker?

We use the Riboruler high range for mRNAs. :)

Good luck!

Hi

It is a late answer, but I hope it add some details.

I work with Gram- bacteria and actually use the RiboPure-Bacteria kit for Total RNA isolation. The kit for itself works very well for total RNA from cultivated bacteria. However, do you know that RiboPure-Bacteria kit for Total RNA isolation is specially designed to lose the small RNA (the filter do not retain the small RNA - this is meant for people who want to continue with rRNA subtraction for RNA sequencing : RNA-seq). If you are interested in “true” total RNA you have to change the kit.

As stated Ward De Spiegelaere, the quality of starting material is very important, and as you use the bacteria from liquid culture to get pellet – this is an important step. When I want to sample bacteria from liquid culture, I collect a wanted volume to the cold tube placed in ice and immediately add 1/9 vol. of ice cold 5% phenol (in EtOH) to stop any transcriptional responses and RNA degradation (if you don’t “block” from the beginning bacterial cells you risk to observe in transcriptomic responses the alteration that have nothing to do with conditions you want to study, like for example cold shock response that appear in bacteria as quickly as in 2-5 minutes). Then, I centrifuge quickly samples, take off supernatant and freeze pellets in liquid nitrogen before to put them at -80°C. The day of extraction I put tubes in ice add the lysis buffer to bacterial cells pellet before thawing. When cells are thawed I can transfer the mix to the kit tube containing beads from the kit and I proceed with 10 min hirizontal vortexing (sometimes I do 15 min). At the and I get RNA of RIN 10 to 9.7 never less.

Concerning the degradation, like stated Ulrike Pfreundt, I confirm that also in some bacteria, like Rhizobium etli, the 23S overcome a fragmentation leading to apparition of two new picks (Vercrusse et al., 2010, BMC genomics 11:53).