Hello RG Hivemind,
For RNAseq of Aspergillus (or other filamentous fungi) with Oxford nanopore MinION, I am wondering:
1. What is the expected percent yield purified mRNA from total RNA? From a cursory search, a range from 1 to 5 % mRNA/total RNA seems standard at a broad taxonomic scale, but what is your experience with fungi?
2. Anyone done PCR-free direct cDNA sequencing with Aspergillus? With low mRNA concentration we may investigate PCR-cDNA sequencing instead.
Some background:
Using liquid N2 mortar-pestle homogenization and Qiagen RNeasy Plant Mini Kit we extracted total RNA from Aspergillus (cultured on liquid media) mycelia with good yield (~300 ng/μl, Qubit IQ integrity = 10).
Using this total RNA as input to the Oxford Nanopore direct cDNA sequencing kit, cDNA synthesis was performed on four input concentrations of total RNA ranging from 0.5 to 5 μg, resulting in a cDNA yield plateauing around 1.75 ng/μl (see attached image; analyzed after RNA degradation and second strand synthesis but before End Prep and Barcode Ligation).
Given that 70 to 200 ng of cDNA are required as input for End Prep and barcode ligation, my cDNA yield is prohibitively low, but perhaps not lower than is expected given the small fraction of mRNA in the input total RNA?
Of course, why not purify the mRNA before cDNA synthesis? Using the NEBNext Poly(A) mRNA Magnetic Isolation Module with 2 μg input total RNA, effectively all mRNA was lost resulting in below 1ng/μl yields of low purity mRNA, if any.
So my choice is between:
(A) troubleshooting mRNA purification until yield is high enough (≥100ng for direct cDNA sequencing or ≥1ng for PCR-cDNA sequencing), or
(B) use ≥50ng total RNA as input for PCR-cDNA sequencing.
I used the NEBNext Poly(A) mRNA Magnetic Isolation Module previously with wasp tissues and had no issue getting good mRNA yield from the insect total RNA. So I am puzzled and open to advice from anyone working in this area :) Thank you in advance for your time and your advice!
- Andrew Legan
Hi Andrew W. Legan , any chance you managed to solve the problem with isolating Aspergillus mRNA using the NEB magnetic isolation kit? We are having the same problem with this kit when trying to isolate mRNA from total RNA of a Penicillium species.
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