Hi Swati,

the A260/280 relates to potential protein impurities, but may also (which is more likely) reflect pheonl that is left over form the Trizol prep. If you are indeed worried about that ratio, try scanning a complete UV spectrum and comapre it to an authentic nucelic acid spectrum. Phenol will show a peak at 270 nm. Of interest is also, that the 260/280 ratio was initially introduced to assess the purity of protein samples contaminated wiht nucleic acids rather than the inverse. Since proteins have fewer chromophores, they show up less clearly, so this parameter is not very precise. Your values, however, a clearly in the green, and if your sample does not show degradation in an agarose gel, you should be in the clear.

Regards, Mark

Hi Swati,

The RNA prepartion parameters looks fine but I'll still suggest you go through the quality requirments required for miRNA deep sequencing....

bye

Hi Swati,

the A260/280 relates to potential protein impurities, but may also (which is more likely) reflect pheonl that is left over form the Trizol prep. If you are indeed worried about that ratio, try scanning a complete UV spectrum and comapre it to an authentic nucelic acid spectrum. Phenol will show a peak at 270 nm. Of interest is also, that the 260/280 ratio was initially introduced to assess the purity of protein samples contaminated wiht nucleic acids rather than the inverse. Since proteins have fewer chromophores, they show up less clearly, so this parameter is not very precise. Your values, however, a clearly in the green, and if your sample does not show degradation in an agarose gel, you should be in the clear.

Regards, Mark

I experienced many times a high absoprtion at 230 when using Trizol reagent. It can influence down stream aplications such as library preparation fore NGS. We never found out why. However, an aditional chlorophorm extraction step followed by a ETOH precipitation helps a lot.

The ratio of 28S to 18S in intact RNA is around 2:1 simply because 28S is around double the size of 18S and incorporates more dye. If the RNA is partially degraded then big fragments are more prone to degradation as they contain more potential cutting sites for RNases and they migrate at different size ranges. As a consequence the ratio is getting lower (1:1 or 0.75:1) or 28S desapears and instead you will see increased signal intensities lower than the 18S. 28S to 18S Ratious of 4 to one I never experienced.

@Stefan

The 230 absorption is probably a shoulder of the phenol peak - supported by the finding that it is removed by an extra chlroform extraction - did you ever measure a full spectrum?

Regards, Mark

Dear Swati,

Your question raised a doubt. I wonder, if you are interested in deep sequencing of small RNA, then, probably, the following step after RNA extraction is the purification of the small fraction of miRNA by acrylamide gel. In the case, which are the inconvenient of some phenol in the RNA samples? Does anyone can give some reasons?

Correctly mentioned by Stefan, lower 260/230 ratio means the sample is likely contaminated with organic chemicals like Phenol and most probably it will hamper your downstream applications where you will be using various enzymes. I have experienced the bitter part of low 260/230 ratio in two of my samples. Better you do a extra chloroform extraction step and surely you can increase the 260/230 ratio otherwise the RIN number that you have mentioned looks very fine.

About 28s/18s ratio if you are able to see two sharp bands and the 28S band is little bit more intense than the 18s you can proceed with the samples. In an idea situation the 28s band should be twice as intense as the 18s.

Hope this clarified your query

Best of luck

@Pedro, Don't you required to do any depletion steps? If yes, do we know the effect of phenol in this particular step?

230/260 ratio of total RNA is not very important if you will extract small RNA fraction from the gel before cloning but the estimate of RNA concentration will be affected to some extent, so reprecipitation is a good Idea (see Stefan's answer) usualy removes the traces of phenol. It is a good custom to wash an RNA precipitate with 75% ethanol before you will dilute it in wateror TE0,1.

28S/18S ratio and RIN should be comparable between samples, then you should get comparable numbers of reads for small RNA molecules derived from rRNA degradation. High and comparable RIN should improve the reproducibility of your assays.

OK, I agree. Wash with ethanol 75% is useful to remove phenol traces, but be careful; it also could contribute to remove the small RNA fraction.

High A230/A260 ratio suggest about residue phenolic compound in your sample. If the ratio is 1.5, it is fine, but of ratio is 0.7, i am not sure you will get good cDNA. Next time your can try to take water-phase more carefully, and, of course, like suggest prevous reseraches, extract with chlorofrom and precipitate with ethanol. However, you may reduced RNA contents. In my hand high A230 come after isolation with old TRIZol reagent. Good luck!

Thanks everyone. I am going to extract RNA again from some of my samples with one extra chloroform extraction and will again post the results (which I pray should be what is expected!)

Hi, Swati, it is good idea to isolate RNA again. Plesae, check TRIzol experience data, old one will give you high A230. Also, be very carefully when take water phase after first cholophorm extraction! Good luck!

Hi, in my experience, to completely eliminate the peak at 230 nm, just repeat the precipitation with isopropanol and washing with 75% EtOH. It works very well, no loss of RNA and is very fast.

Hi Lufti

Yes I did two extractions with cholroform and got pretty improved 280/230 ratios. In addition to this I did, the precipitation step on ice with chilled isopropanol. To the pellet I added chilled 75% ethanol, vortexed it ( so that the pellet is thoroughly washed) and then centrifuged at 15000g for 15 min.

Hi Lufti

Yes I did two extractions with cholroform and got pretty improved 280/230 ratios. In addition to this I did, the precipitation step on ice with chilled isopropanol. To the pellet I added chilled 75% ethanol, vortexed it ( so that the pellet is thoroughly washed) and then centrifuged at 15000g for 15 min.

Sorry someone else was logged on Research gate..didn't realized until I posted the answer.

Hi Louis

You take the aqueous layer and add chloroform to it, shake it vigorously for about a min, let it stand at room temperature for 10min and then centrifuge at 12000 rpm (appx).

Hope it helps.

I was wondering why do you need to let your sample incubate at RT 5 min after putting it in a new tube ? (step 1)

What if my sample was stocked in Trizol at -80°C ? The thaw isn't sufficient for the lysis ?

Thanks a lot for your answers

We should see two sharp band on the gel (18S and 28S rRNA).

Why the 28S is more sharper than 18S?

where are the band of mRNAs?

Thanks

Has anyone used phase lock tubes after addition of chloroform, prior to spinning? Also, how much chloroform do you add if you're doing that step twice? Does it depend on the volume of your aqueous phase?

HI Everyone. Pls, what is the expected size of 28S and 18S RNA after running on horizontal agarose gel electrophoresis ???

Hi! Mohammed

Around 3000 bp and 2000 bp respectively.

Hi Swati

Some papers in attachment 1

https://jxb.oxfordjournals.org/content/63/17/6069.short

2

https://virologyj.biomedcentral.com/articles/10.1186/s12985-015-0376-3

3

https://virologyj.biomedcentral.com/articles/10.1186/s12985-015-0376-3

4

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4921785/

5

https://www.sciencedirect.com/science/article/pii/S0888754310001953

6

https://www.cd-genomics.com/Metagenomis/16s-Sequencing

7

https://www.qiagen.com/mx/resources/faq?id=c59936fb-4f1e-4191-9c16-ff083cb24574&lang=en

8

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=10&cad=rja&uact=8&ved=0ahUKEwiq0IOavqLTAhWM7BQKHaGwCFMQFghjMAk&url=https%3A%2F%2Frepositorio.cespu.pt%2Fbitstream%2Fhandle%2F20.500.11816%2F201%2FTese%2520definitiva.pdf%3Fsequence%3D1&usg=AFQjCNHrn4xq-dJ0yYKmY0WxWhC_LuRwFA&sig2=aiQo3nScRxQnse4jHFxoRQ

9

https://pubs.rsc.org/EN/content/articlehtml/2017/mb/c6mb00699j

10

https://www.labome.com/method/RNA-Extraction.html

11

https://journals.plos.org/plosone/article/asset?id=10.1371/journal.pone.0148260.PDF

Good Luck