University of Groningen RNA regulation in Lactococcus lactis van der Meulen, Sjoerd Bouwe

(1)

University of Groningen

RNA regulation in Lactococcus lactis

van der Meulen, Sjoerd Bouwe

IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below.

Document Version

Publisher's PDF, also known as Version of record

Publication date: 2018

Link to publication in University of Groningen/UMCG research database

Citation for published version (APA):

van der Meulen, S. B. (2018). RNA regulation in Lactococcus lactis. Rijksuniversiteit Groningen.

Copyright

Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).

Take-down policy

If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.

Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum.

(2)

525070-L-bw-Meulen 525070-L-bw-Meulen 525070-L-bw-Meulen 525070-L-bw-Meulen Processed on: 24-10-2018 Processed on: 24-10-2018 Processed on: 24-10-2018

Processed on: 24-10-2018 PDF page: 159PDF page: 159PDF page: 159PDF page: 159

(3)

Processed on: 24-10-2018 PDF page: 160PDF page: 160PDF page: 160PDF page: 160 160

Addendum

Bleach as a replacement for urea in polyacrylamide gels used for Northern

hybridization

Detection of RNAs is routinely done by Northern hybridization. Small- or fragmented RNAs are typically separated via electrophoresis through polyacrylamide (PAA) gels containing urea. In Chapter 4 and 5 we have shown that urea can be replaced by a low amount of common household bleach (5% sodium hypochlorite) to denature secondary RNA structures and inactivate potential RNases. PAA-bleach gels can be stored in the cold, allow easy sample loading and reduce the use of chemicals.

Recent advances in RNA sequencing have resulted in the discovery of numerous novel RNAs from various biological origins, including small non-coding regulatory RNAs such as microRNAs from eukaryotic organisms and small regulatory RNAs from prokaryotes. These novel RNAs are validated using several methods, but mostly by RT-PCR and classical Northern hybridization(1). Although RT-PCR is faster, Northern analysis gives insight in the size of the RNA examined and can give a view of possible RNA breakdown. It furthermore has a high specificity and the expression of RNAs can be compared between RNA samples on a single blot. Altogether, Northern hybridization is a widely applied and important molecular tool for studying RNA molecules.

For Northern hybridization, total RNA is isolated and size-separated using 8 to 12% (depending on the size of the probed RNA) PAA gels under denaturing conditions. The RNAs are subsequently transferred onto a membrane to which they are cross-linked using UV light or a heat treatment. A chemiluminescent or radioactively (32_{P) labeled DNA probe is then}

used to selectively bind to the RNA on the blot, which is detected on an X-ray film or using a phosphor screen. PAA gels usually contain 7 M urea or commercially equivalent products as a denaturing agent. Urea forms multiple hydrogen bonds with the bases in RNA, which prevents secondary structures from forming in the RNA. However, the high concentration of urea confronts researchers with a number of inconveniences. For example, polymerized PAA gels cannot be stored at temperatures below room temperature as the urea would then crystallize. Second, during RNA loading, the urea quickly migrates from the PAA matrix, leading to a high concentration of urea in the wells. The wells need to be flushed prior to sample loading, to keep the RNA in the wells before applying a current.

Bleach was identified by Aranda et al. as an alternative denaturing agent, based on its

successful application in agarose gels to evaluate the overall RNA quality by assessing the major ribosomal RNA ratios(2). We show that 7 M of urea, which represents approximately 42% w/v of the composition of the PAA gel, can be completely replaced by adding 1% commercial household bleach immediately prior to gel polymerization. This resulting 0.05% sodium hypochlorite performs equally well in preventing RNAs from folding, which would otherwise lead to smears and fuzzy bands. Furthermore, the low percentage of bleach

(4)

Processed on: 24-10-2018 PDF page: 161PDF page: 161PDF page: 161PDF page: 161 161

Addendum

is capable of inactivating RNases, as has been shown earlier in an agarose matrix(2). We successfully replaced urea by bleach for all Northern hybridization experiments reported in Chapters 4 and 5.

Since Northern hybridization is widely applied in molecular biology, and nearly all protocols rely on the same composition of the PAA gels, we believe this modification is a highly convenient adjustment of the method. It should be an advantage to many researchers, in particular those who study small non-coding/regulatory (micro)RNAs. We also foresee further application of PAA-bleach gels, for instance in primer extension analysis.

REFERENCES

1. Alwine JC, Kemp DJ, Stark GR. 1977. Method for detection of specific RNAs in agarose gels by

transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc Natl Acad Sci U S A 74:5350-5354.

2. Aranda PS, LaJoie DM, Jorcyk CL. 2012. Bleach gel: a simple agarose gel for analyzing RNA quality.

(5)