Hi researchers,
I want to do RNAseq using the flow sorted cells from mouse retinae tissue. The RNA is the pool of many samples and the total yield is between 50-100ng and two samples have 5.5 RIN value. Although the bioanalyzer gel picture is okay and showing good rRNA bands. My understanding is that with this limitations I should go with rRNA depletion. I need experts advise that should I go with the poly A capture or rRNA depletion? Please share a good comparative research article if possible.
"total yield is between 50-100ng", this is clear enough but still I want to confirm this is not the per microliter values you are mentioning.
I don't work with the eukaryotic cells, however, I have experienced in rRNA depletion and may be polyA capture, one can expect 10% recovery and 90% loss of the start material. The kits themselves prefer to start at least 1µg (1000 ng) as a start material.
With concentration as low as yours, I would say this is an indication that you need to stop and reconsider your sampling and extraction and may be sequencing scheme too before moving forwar. By starting with such ow concentration, if you even get some barely workable concentration, once can not rely on the depth and coverage of the signals in the sample. With signal I mean the transcripts and any other RNA form which you might be interested in.
May be you can consult with experts or people in your sequencing facility and they might help you find a best way to deal with such problems.
Hi Abhijeet,
50-100 ng is the total yield. Illumina rRNA depletion kit input is ~25ng (illumina standard ribo zero plus kit cat# 20040525). Its really difficult to get good yield with the sample I am dealing with. I am discussing with the application scientist now. My main concern is poly-A capture vs rRNA depletion library preparation should not cause differential coverage for DEGs.
Apart from your concentration problems, if just the methods are to be considered, I don't think this would be a problem as long as you treat all the samples in the same way. Further you can also consider to go through the manual of kits you will be using and look for the efficiency and reliability. Illumina kits are good for rRNA depletion and I don't have much idea about polyA capture kits. Your application scientist might come up with some solution. In that case it would be nice if you update your solution here, it might help others.
Hi Abhi,
I think with two samples, 5.5 RIN is ok and your samples should be acquired fine by the company. I remember my 1-2 samples had a RIN value like that and turned out to be fine during comparison. Since your RNA amount is low, best is to send it like this and acquire them. I won't play with them more because that would deplete RNA more as Abhijeet suggested above. Sometimes RIN is also misrepresented-
Please also read this answer here
https://www.researchgate.net/post/Which-value-of-RNA-Integrity-Number-RIN-is-considered-suitable-sufficient-for-RNASeq-analysis
Hi Abhi
Since RIN number is quite low (still acceptable), and RNA concentration is also low, better do not play much and take consultation from the genomic facility. They will be able to support you with the insights as well as best possible method to get the best results. Dr. Lamb at UPITT genomic facility might be helpful.
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