Hi everyone,
I just recently did some RNA extraction for my insects sample for RNA-seq. I have checked both the concentration (average 450ng/uL) and 260/280( ~2.0). I also done a agarose gel run to check the integrity of the RNA but was't sure if they are considered good quality RNA or degraded as they is 2 distinct band 28S and 16S observed in the gel but with some smear
I think the quality is really good. Smear is very common but you can proceed further experiments with this RNA. Do not freeze and thaw.
As you don't mention what you used to extract this RNA from, but whatever you used (seems like some bacteria), you did a good job in your protocol.
Furthermore, two bands and smear:
Normally, doing gels for RNA is (in my opinion), the waste to RNA samples. I would prefer a bioanalyzer quantification to gel. Nevertheless, as you mentioned there are two distinct bands of 23S and 16S. You are right. And one should expect a smear in RNA gels. The reason being, there are hundreds of different RNA molecules of different sizes in any cell. Thus, if you try to visualize all of these molecules, they will be separated based on their size/mass. This will cause a smear. So, in case of RNA gel "smears are good".
RNA integrity is quantified in terms of RIN or RQS. Gel electrophoresis is an old but sufficient technique to know the integrity of the RNA, but you can't certainly say to what extent the RNA is integrated. If you use a bioanalyser for RNA (Either total or small RNAs), you will get RQS score, from 1 to 10. 1 means degraded while 10 means highly integrated RNAs. Bioanalyser is a sophisticated electrophoresis instrument, which calculates RQS or RIN based on 23 and 16S RNAs in the sample, like you can see in your gel (which are highly stained). However, some downstream techniques like transcriptome and miRNome analysis require to check the integrity of the samples, which requires Bioanalysis of RNAs. Nevertheless, your sample shows that it is good in integrity, but to which extent, is difficult to tell!
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