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!
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!
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