we are trying to obtain RNA from nuclei and RINs are very low no matter what we have tried. ...Should the RIN be used? is there another method to assess the quality of the nuclei RNA?
THANKS
Dear Azucena,
RIN is the most widely used score used to evaluate RNA integrity. There are commercially available kits that provide the RIN value of a particular sample using only 1ul or less starting material.
Lower RIN values indicate your RNA samples are most probably fragmented. We routinely use RNeasy Minikit from Qiagen for RNA extraction to extract RNA from cell lines and human tissue and usually have received RIN values >7.
I hope this helps.
Best,
Ayan
Hi Azucena Salas
Could let please let us know the Nanodrop 260/280? & RIN values of your samples?
Regards
Saurabh
You might find this recent work by Cholet et al useful which discusses using RIN and rAmp Article Differential ratio amplicons (R amp ) for the evaluation of ...
Dear all, thanks for your answers. We do use RIN regularly for whole cell RNA, but my question is about NUCLEAR RNA. In that case, i would expect to find larger RNA molecules and not necessarily the 18S and 28S, but 32S or even 45S.
Azucena Salas did you find you answer yet? I have been running Bioanalyzer chips with nuclear RNA and I cannot find a reference to check for the quality.
Steven Francis Grieco hey what's up? How are your samples looking like? Can you upload an example? I have very few nuRNA and I cannot find a reference for this either.
Maybe running the nucleus RNA in gel can help check the quality, like figures shown in this document
Qingtao hu thanks for your answer. But in my case that wouldn't work as I have picograms of RNA and I cannot use it all to run it in a gel.
Francisco Javier Rodríguez Baena hi, what is your purpose of the nuclear RNA? For using in single cell RNA sequencing, the technical support from 10x Genomics suggests that an RT and amplication of cDNA using SMARTseq method and then running on 2100 to check the length distribution. Usually, the peak among 1-2k bp is OK for the scRNAseq.
Azucena Slas, I agree that RIN which measures 28S and 18S is not a good measure for RNA. I am facing the same problem. I need a good method to know whether the nucleus is fit for 10x or not.
Hello Debdatta Halder Banerjee that where I am right now, not 10X but normal RNAseq which is basically the same. And still haven't got a clear answer. For the 10X you could always to the library prep yourself, do cDNA with one or a couple samples and the if the amplification has worked, but still no quality checks
Francisco Javier Rodríguez Baena thanks for the suggestion but the issue is that once I start preparing the library and go till QC1, that's a point of no return. The chip has already been used so might as well finish the whole prep. Are you suggesting me to do something else? In that case I might have misunderstood you.
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