total RNA isolation: miRNeasy kit vs trizol

[Chinanicole]

I am isolating total RNA from a small number of drosophila heads (20) for diff gene expression analysis on NextSeq500. My goal is to generate two separate libraries from the samples: 1) coding RNA via poly(A) selection from total RNA and 2) small RNA library (<200 nt). I tried using the miRNesy micro kit from Qiagen (which in theory, allows for separation of a small [<200 nt] and large [>200 nt] fraction) however, I kept getting rRNA in my small RNA fraction and low yield in the large fraction. Qiagen was not helpful in trouble shooting so I moved away from kits entirely and have been optimizing isolating RNA from heads by trizol/chloroform with in solution DNAse digestion.

Total RNA isolated by trizol/chloroform+ DNAse digestion has been QC'd via bioanalyzer and the gel and it appears there is no degradation of rRNA so I'm fairly comfortable moving forward with using trizol-extracted RNA for my poly-A(+) library prep. Any reason to think my sample might not be pure? Any concerns with not using column based?

Found this paper Sultan, M. et al. Influence of RNA extraction methods and library selection schemes on RNA-seq data. BMC Genomics 15, 675 (2014). which makes it seem like trizol/chloroform is comparable to Qiagen columns.

Thanks!

[nucacidhunter]

There is no particular issue using Trizol. You should check 260/230 and 260/280 ratios to ensure purity.

Most of small RNA-seq library prep kits can use total RNA as input (prefered input) so there is no need to isolate small RNA.

[huguesparri]

Just be aware that in drosophila, 2S RNA is abundant and 30nt long.
You may (will?) need a specific protocol to build smallRNAseq libraries from drosophila RNA.

[kmcarr]

Just be aware that in drosophila, 2S RNA is abundant and 30nt long.
You may (will?) need a specific protocol to build smallRNAseq libraries from drosophila RNA.

This is VERY important. We did a miRNA-Seq project for a client from Drosophila and were not aware of this fact. Output reads were 70-90% 2S rRNA depending on library. There is a specific protocol which uses a blocking oligo to prevent reverse transcription of the 2S rRNA. We have not tested this in our lab so cannot give any advice or insight as to how well it works.

[Chinanicole]

Thank you all! I was planning on isolating the relevant small RNAs from total RNA by gel but wasn't aware that the 2S unit migrated so closely to miRNA/piRNA. Will check out the protocol, thanks!

[Chinanicole]

Just a quick follow up - I am NOT doing a separate column based clean up of the total RNA isolated by trizol/chloroform. Because I am using so little starting material (20 fly heads) - the column just diminishes my RNA yield. Any concern? Thanks!