Hi all, I am working with small RNA fragments (30-50 bases).
After ribosomal RNA depletion, I am exausting almost all of the RNA and there seem to be almost negligible yield of RNA (starting input RNA is 50 ng) and kit used to deplete rRNA is Ribozero. What would be the best way to improve the yield after rRNA depletion, should I increase the starting material?
Hi Mansi,
I have not encountered your exact problem, but there are few common suggestions I can make. First of all, increasing starting material is always a good strategy to get better sequencing results (within the kit input range, of course). Secondly, you may revise your bead cleanup steps: 30-50bp fragments are really small, and usual 0.8x or even 1x beads may cut them off. So, higher proportion of beads may increase the yield of such small fragments (1.2x-2x I assume). And finally, you can first do short initial PCR amplification, and only afterwards do the rRNA depletion (like in Takara SMARTer Pico mammalian kit, for example). It is specifically designed for low inputs, and it is whole transcriptome. First you reverse transcribe everything with random priming, then you fragment the cDNA and amplify all the fragments, and only afterwards you deplete fragments, which relate to rRNA (something like CRISPR-Cas9 with rRNA-related probes as guides).
https://www.takarabio.com/learning-centers/next-generation-sequencing/technical-notes/rna-seq/stranded-libraries-from-picogram-input-total-rna-(v2)
Good luck,
Michail
Dear Mansi, rRNAs are just an abundant (80%), but actually non-coding parts, for which you no need to worry about much. In my opinion and experience you can omit this step regarding using any Ribo Zero sort of protocols.
Actual problem lies in the convoluted tertiary structures of RNA like presence of peudo knots, kissing hairpins and hairpin loops, which may hinder the production of a fully length cDNA strand unless not tackled properly well before the initiation of reverse transcription.
This can be handled by denaturing total RNA content for 65 C for 2 minutes then place the samples on ice and proceed to RT, although many cDNA kits already have this RNA-linearizing strategy builtin. Often people use up to 10% DMSO in RT reactions to ease this issue of RNA complexity.
A standard poly A tailing protocol is often adopted on final RNA yield to increase the content of mRNA, if this is feasible to you.
The best solution is to introduce maximum initial total RNA content in RT reaction, which is at least 1 ug of RNA (though if more it would be ideal).
Then comes the choice of RT enzyme, which must have an increase processivity and fidelity and can withhold even high temperatures (above 50 C) and can also generate near or above 9-10 kb cDNA full-length transcripts. For this, I always prefer SSIV enzyme containing cDNA kits, their results are unimaginable for all kind of samples. And then the choice of RT primers and the best is comixture of Oligo DTs and random hexamers, as together both increases the sensitivity of an assay remarkably. cDNA kit using any primer alone will not cover all the available transcripts as broken mRNA fragments can be missed here and gDNA can also be amplified in case of missing Oligo DTs, as well as using an increased concentration of random hexamers, longer cDNA fragments will not generate much.
I highly advise you to use fluorimetric approach to quantify your RNA like Qubit.
And carefully focus on RNA extraction protocol of your kit as well. Use the proper kit and modify the protocol as per your sample type.
Sincere regards....
Hi David,
Thanks for your answer. Can you suggest me how much starting material (RNA) should I begin depletion with? Should I go higher than 1ug. The limitation with my experiments is, small RNA I generate have very low yields. Though, I am trying to increase my cell number to 100 million.
David Farringdon Spencer
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