Hi all,
I try to figure out weather there is a change in my rRNA following treatment.
when I treat the cells (mouse cells) and isolated RNA, I see a major difference in RNA concentration, without any difference in cell number.
what is the best way (or easy for start) to see if there is any change in the ribosomal RNA amount, activity or biosensis?
Thanks,
Shani
Roughly 80% of the total RNA consists of rRNA and so if your treatment causes a decrease in total RNA, then it is highly likely to decrease rRNA synthesis. To a lesser extent, the decrease in RNA content could be due to RNA degradation perhaps via ribophagy. Decreases in rRNA synthesis can be seen easily enough. You can use fibrillarin staining to look at nucleolar size and decrease in nucleolar size constitutes a decrease in rRNA synthesis, as nucleoli are formed on the sites of rRNA synthesis - the rDNA. If you want to quantify the decrease, you can do so using quantitative real time PCR. You can generate primers against the External Transcribed Spacer (ETS) or the Internal Transcribed Spacer (ITS) of the 45s pre-rRNA. The ETS and ITS are spacers that are cleaved during rRNA biogenesis and are degraded subsequently, and therefore a decrease in pre-rRNA would be indicative of decreased rRNA synthesis. You can perform qRT-PCR and use your ETS and/or ITS primers to estimate decreases in rRNA biosynthesis.
Thank you Sabarish!
Actually, my treatment cause to increase in Rna concetration, but I guess the idea is the same?
Can bioanalyzer can tell me something about rRNA amount?
Yeah, increase in RNA would most likely lead to increased nucleolar size, and it is easy enough to see using a nucleolar marker. Also, ETS and ITS levels are indeed indicative of both positive and negative changes in rRNA levels. But, you have to use the proper control(s) such as tubulin or GAPDH, which are not influenced by your treatment.
Thank you Sabarish Nagarajan !
Maybe you have primers for mouse ITS or ETS? that will be very helpful
Sorry, I worked mostly on fruit flies. Just google it up. It shouldn't be too hard to find. Most people working on ribosome biogenesis and cell growth should have used them. I may have some papers at home, I'll forward them if you can't find any.
@Matthias R. Schaefer: GAPDH and beta-tubulin are just examples of housekeeping genes, but they may change depending on the treatment. You have to perform pilot studies using equally seeded, treated vs untreated cells and study the expression of 3-10 different housekeeping genes and perhaps even ribosomal mRNAs (eg. rps9, rpl20). You can manually choose 2-3 which are stable even after your treatment. Alternatively you can use software such as geNorm or qbase+ software from biogazelle.com. Using 2 or 3 reference mRNAs is better than choosing a single mRNA for reference. For more details please refer to this: http://www.idtdna.com/pages/support/technical-vault/video-library/real-time-pcr
Hi, sorry to jump into the discussion so late. I was wondering if you all think a similar approach would work to investigate ribosome biogenesis a little more in depth? I have a model that maybe impacts ribosome biogenesis and I am interested in knowing at what level of the processing this happens. Do you think it is possible to design primers for the complete repeating unit, ETS and ITS and look at the different levels of expression?
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