I have isolated total RNA from mammalian cell culture samples. I used a Qiagen Kit. I have checked the conc and quality of RNA by Nanodrop. Ratio of 260/280 was between 2.01 and 2.09, and 260/230 was between 2.1 and 2.26. However when I checked the samples for RIN (by Agilent technologies), the RNA samples didn't look good. RIN number for a few samples were between 3-6 and few samples were between 7-9. However, all samples looked good on Nanodrop. Now I dont understand, whether there is any problem in Nanodrop reading or RIN quantification by Agilent technologies.
RIN is based on a much more comprehensive assessment than a NanoDrop reading. NanoDrop provides concentration and purity; that is, whether small contaminants are present. It will not indicate intactness of RNA (like whether you have full-length rRNA or just fragments), and DNA will be measured along with RNA. I don't know all details of the RIN algorithm (it's proprietary), but it takes into account multiple features along the "electropherogram." It might be worth pointing out that there's no magic cut-off for good and bad RIN. RIN>7 is generally accepted as decent RNA, but for some applications, it would be too low. RIN of 3 is generally considered poor quality, but if your goal is amplifying short amplicons (including miRNA), it might be usable.
RIN is based on a much more comprehensive assessment than a NanoDrop reading. NanoDrop provides concentration and purity; that is, whether small contaminants are present. It will not indicate intactness of RNA (like whether you have full-length rRNA or just fragments), and DNA will be measured along with RNA. I don't know all details of the RIN algorithm (it's proprietary), but it takes into account multiple features along the "electropherogram." It might be worth pointing out that there's no magic cut-off for good and bad RIN. RIN>7 is generally accepted as decent RNA, but for some applications, it would be too low. RIN of 3 is generally considered poor quality, but if your goal is amplifying short amplicons (including miRNA), it might be usable.
It is interesting that you report extracting RNA from tissue culture cells, which usually yield good quality RNA. Are you using trypsin prior to your RNA extraction? If so, it is perfectly possible to get RNA that looks acceptable by the usual ratio criteria but is in fact totally degraded. I am attaching a slide I show when I give my talks, data obtained by Peter Vrtačnik from the University of Ljubljana in Slovenia.
In any event, Kenneth's answer is a good one and you could use a 3':5' assay (as described in our Nature Protocols paper) to obtain an additional and directly relevant quality assessment of your samples.
Nanodrop reading doesn't saying anything about degraded RNA..
Nanodrop only give the total material that absorbe at 260 and 280 nm, se have not the qualité of sample integrity of sample.
The nanodrope give us the quantity yeald and the pourcentage of contamination with proton (260/280). THE integrity of THE sample only gave by migration on agarose gel or bioanalyseur.
Nanodrop is not completely reliable to access your RNA for a particular application. In one of our experiments we tried extracting RNA from oocytes which were matured in vitro.Nanodrop shows a very good reading but upon real time PCR analysis the results found to be very poor.here we did not do any trypsinization.
Nanodrop tells you about the purity of RNA and concentration, while RIN value is all about the quality of RNA i.e whether it is intact or degraded.
Although we do not know the RIN algorithm used by the Agilent 2100 Bioanalyzer it must be based on densitometry of gel electrophoresis of total RNA samples. That is, the peak area of the 28S rRNA should be approx twice the peak area of the 18S rRNA (in the case of animal cell total RNA). For plants or bacteria the principle is the same. If we observe this ratio, we consider that the preparation of RNA is of good quality. We'd make it years ago when there was not the Bioanalyzer. What this company did was to create an algorithm to calculate this automatically. In conclusion, the Bioanalyzer is an apparatus/method to evaluate the integrity of RNA samples. The NanoDrop is to quantify nucleic acids in a sample based on spectrophotometry rules.
Depends on your application.......
If your aim is Realtime PCR,
Then simply run your total RNA on Formaldehyde agarose gel and check integrity. You should get two sharp bands first one 28 S rRNA which should have double size of second band 18srRNA. We can assume it as 2:1 ratio but bands should be sharp.
NanoDrop will only gives concentration of RNA not integrity
Nanodrope gives only an idea onto the purity of the product and the quantity while Bioanalysieur (RIN) allows to give the quality of the product and its integrity.
These 2 instruments work in complementation to have the quantity and the quality of ARN's production.
I agree with Samira, and note that NanoDrop serves to quantify the RNA samples and the Agilent Bioanalyzer serves to make microfluidic electrophoresis and therefore to analyze the integrity of the samples. Are two different methods for different purposes. This means that a sample can have very good UV absorbance ratios, but still being degraded. For this reason we have to do the analysis of RNA samples by agarose gel (conventional) or via Agilent Bioanalyzer.
RIN number of my sample has come 7.7 and I am going to perform small RNA sequencing using proton torrent platform Kindly give me your inputs asap
Kindly find attached graph
Hello to everyone,
this is what happens to me:
- I have comparable good concentrations and qualities of all my 12 RNA samples, measured by Nanodrop.
- Strangely, only 2/12 have no RIN. The Bioanalyzer says that the RNA is not detectable. I have run them both in a diluted and undiluted way and again it tells me that the RNA is not detectable. This is very strange because I have great RNA concentration for the Nanodrop. Also, the concentration measured by the Bioanalyzer for both the diluted and undiluted RNAs does not fit with the dilution that I did and of course is very low (is it normal?)
- All the 12 samples have been treated in the same way (all of them have been treated with Trypsin)
Any advices and explanations?
Thank u very much in advance!
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