Good RIN, bad mRNA?

SeqVicious · 12-11-2014, 01:05 PM

Hello, I'm preparing some RNA for RNAseq and I submitted my samples to the local sequencing core for quality control on the bioanalyzer. The results came back and my samples have good RIN values, almost all >9. But I can't see a nice bump for the mRNA in which I am interested. I'm not sure if all of the peaks along the electrophoretogram are obscuring the mRNA. Should I trust these samples based on the RIN value alone? I want to proceed to ribosome depletion but I don't want to waste an expensive kit on samples that might not have good mRNA in them.

The attached is an example of my bioanalyzer results.

Thanks!

MU Core · 12-12-2014, 03:26 PM

You won't visualize the mRNA using the standard RNA quality check. The mRNA is a small percentage of the total RNA from the extraction. What is being visualized is the ribosomal RNA which when intact, as in your sample, you infer a similar level of quality to the mRNA in the sample.

Asaf · 12-14-2014, 12:34 AM

If you're still skeptic you can always take some of the RNA and PCR your mRNA of interest to make sure it's there. However, you shouldn't worry as MU Core said, there are no peaks for mRNAs in bioAnalyzer.

kerplunk412 · 12-16-2014, 04:17 PM

mRNA is only 1-3% of total RNA, so as others have said you can't really see it at all in total RNA. Having intact rRNA peaks is basically an indication that you are not seeing significant degradation in your sample, so you can safely assume that your mRNAs are also intact.

I will also save you some confusion later: after rRNA depletion you have a large peak of tRNA, so you STILL can't see the mRNA. Also, since so much of the rRNA depleted sample is tRNA, you will need to use more rRNA depleted sample vs poly(A) enriched sample for library prep. For example, if your protocol calls for 1 ng of poly(A) RNA, you should probably use at lease 10 ng of rRNA depleted RNA.

Also, since you are performing rRNA depletion it doesn't matter much if your RNA is somewhat degraded, as long as it isn't very badly degraded. So I think you can confidently proceed with the RNA you have. Good luck!

SeqVicious · 01-05-2015, 03:20 PM

Thank you for the responses. I will trust my RNA based on the rRNA peaks and continue on!

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12-12-2014, 03:26 PM	#2
MU Core Member Location: Columbia, Missouri Join Date: Apr 2008 Posts: 57	You won't visualize the mRNA using the standard RNA quality check. The mRNA is a small percentage of the total RNA from the extraction. What is being visualized is the ribosomal RNA which when intact, as in your sample, you infer a similar level of quality to the mRNA in the sample.

12-14-2014, 12:34 AM	#3
Asaf Member Location: Jerusalem Join Date: Jul 2014 Posts: 20	If you're still skeptic you can always take some of the RNA and PCR your mRNA of interest to make sure it's there. However, you shouldn't worry as MU Core said, there are no peaks for mRNAs in bioAnalyzer.

12-16-2014, 04:17 PM	#4
kerplunk412 Senior Member Location: Bioo Scientific, Austin, TX, USA Join Date: Jun 2012 Posts: 119	mRNA is only 1-3% of total RNA, so as others have said you can't really see it at all in total RNA. Having intact rRNA peaks is basically an indication that you are not seeing significant degradation in your sample, so you can safely assume that your mRNAs are also intact. I will also save you some confusion later: after rRNA depletion you have a large peak of tRNA, so you STILL can't see the mRNA. Also, since so much of the rRNA depleted sample is tRNA, you will need to use more rRNA depleted sample vs poly(A) enriched sample for library prep. For example, if your protocol calls for 1 ng of poly(A) RNA, you should probably use at lease 10 ng of rRNA depleted RNA. Also, since you are performing rRNA depletion it doesn't matter much if your RNA is somewhat degraded, as long as it isn't very badly degraded. So I think you can confidently proceed with the RNA you have. Good luck!

01-05-2015, 03:20 PM	#5
SeqVicious Member Location: New Hampshire Join Date: Sep 2011 Posts: 20	Thank you for the responses. I will trust my RNA based on the rRNA peaks and continue on!