Hello Shruti,

the answer is basically already there, in your question: if you synthesized your cDNA from total RNA, still containing rRNA (i.e. no depletion), your cDNA can be, in line of principle, sequenced using an Illumina platform, however a huge fraction of your reads will map to ribosomal RNA, which is something you should definitely avoid. So, if RNA from your sample(s) is still available, you should either

1) Purify using Oligo-dT technique

PRO: you don't need to further ribo-deplete and you can go straight to quality control and lib-prep

CON: Oligo-dT is inefficient in presence of degraded RNA. If this is the case you may end-up with a significant decrease in the reads counts of 5'-proximal regions, due to loss of mRNA that is physically distant from poly-A.

2) Perform ribo-depletion from total RNA and then proceed with cDNA synthesys from ribo-depleted total RNA

PRO: more robust (up to a certain extent) if RNA is partially degraded

CON:

- you have to buy the ribo - kit

- depletion is never 100% efficient so you may still found a significant number of reads mapping to rRNA.

Hi Shruti,

Assuming you have had successful during first strand synthesis of cDNA, you must synthesize the second strand to stabilize youd DNA sample. ssDNA is not quite estable and it is prone to be degraded even upon freeze storage. Once you got the second strand synthesis (with E.coli DNA Polymerase I, E. coli DNA Ligase, RNase H, dNTP's, and T4 DNA Polymerase) you could purify ds-cDNA by any column protocol and then send your sample to be sequenced. Although as you mentioned, with not rRNA depletion you will get aprx 90% of your reads matching against these molecules.

Good luck,

Thank you for your replies Drs. Piazza and Benitez-Paez. However, if for whatever reason I do NOT have any remaining RNA would you say it would be a waste of time (and money) to sequence the cDNA as is due to the low number of mRNA-specific reads?

Would a microarray approach be more useful then? (at the cost of losing out on some data)

Thanks!

Yes, I would say so.

Unless the sample is extremely valuable for you I suggest not to proceed. However, if you decide to go on with the sequence, consider that mRNA is approximately 2% of the total, therefore you will need a very high number of reads to get a good sequencing coverage.

In my point of view, the best thing you can do ( if you can ) is to perform a new RNA extraction. And I totally agree with Alfonso Benitez-Paez: you have to stabilize your cDNA sample with a double strand step before going to Illumina sequencing.

Shruti,

Depending on your budget, you can also just go ahead and sequence your sample (after finishing your library) with a high sampling depth like suggested by Dr Piazza. You will still have a tremendous amount of ribosomal material but that way, you might have enough mRNA reads for your analysis.

I am attaching a paper on how to remove rRNA, if you're interested, for your next project.

thank you all for your replies. Based on your feedback, I've decided to abandon this project until I can repeat the isolation process and make sure I ribo deplete this time.

However, just for discussions sake, Thermo has a RiboMinus™ kit (https://www.thermofisher.com/in/en/home/life-science/dna-rna-purification-analysis/rna-extraction/rna-applications/ribosomal-rna-depletion/ribominustechnologypage.html) that eliminates rRNA by using rRNA specific probes. My question is if these probes will not be useful once RNA is converted to cDNA. The website says "..does not affect the ability of the oligonucleotide to bind DNA or RNA". In that case, shouldn't this potentially be applicable to clean up whole cellular cDNA as well?