Can I use this RNA smaple for RNA-seq

[Ken_HK]

Hi, I have a RNA sample from Trizol RNA extraction with Bioanalyzer QC 28s/18s:1.8 and RIN 9.4. However, there is a high peak below 200nt. Can I use this sample for RNA-seq? Pls also see the attached RNA profile

[pmiguel]

Hi, I have a RNA sample from Trizol RNA extraction with Bioanalyzer QC 28s/18s:1.8 and RIN 9.4. However, there is a high peak below 200nt. Can I use this sample for RNA-seq? Pls also see the attached RNA profile

Sure, your RNA looks great. Trizol preps normally do not remove the small RNAs abundant in all cells. That is what you are seeing below 200 nt.

The only caveat I can think of is that if you are doing standard RNAseq, input amounts of RNA may not be calibrated for large percentages of the total RNA prep being small RNAs. It should not present a problem in your case, but I have seen preps where it looked like >70% of the RNA was small.

If you are concern there are lots of methods to fractionate away the small RNAs. For example Zymo RNA clean up columns come with a method for washing off small RNA prior to eluting.

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Phillip

[Ken_HK]

Thx for your answer. But do you think is there any chance that those peaks are DNA fragments? Moreover, I used the RNA 6000 pico kit. Will that be more sensitive to small fragments compare with nano kit and that's why I have a high cluster of peaks below 200nt?

[pmiguel]

Sure, they could be DNA fragments -- but to me this looks like a normal Trizol extracted RNA chromatogram. Overall pico chips are more sensitive than nano chips, obviously. But I have not noticed pico chips being unusually sensitive to shorter molecules.

Again, if the low molecular weight stuff is really bugging you, just get rid of it.

--
Phillip

[SamCurt]

I agree with pmiguel; this is typical of TRIzol extractions. Because I usually think DNAse treatment should remove all of the gDNA in the sample if used as directed, I won't worry much about gDNA contamination.

Of course pasing through a column would remove this peak, but this can also be done by isopropanol precipitation using a lower volume of isopropanol relative to the aqueous phase.