Question: Why Do You Need To Use Cdna For Rna-Seq?
15
gravatar for Justin
6.5 years ago by
Justin440
United States
Justin440 wrote:

Why do RNA-Seq protocols involve reverse transcription of RNA into cDNA and sequencing cDNA? What are the problems with sequencing RNA directly?

Are there problems with doing reverse transcription? The wiki page on RNA-Seq says, without citation, that

converting RNA into cDNA using reverse transcriptase been shown to introduce biases and artifacts that may interfere with both the proper characterization and quantification of transcripts

Also, is the cDNA single-stranded or double-stranded?

cdna rna-seq • 32k views
ADD COMMENTlink modified 2.7 years ago by SeqFreak0 • written 6.5 years ago by Justin440
39
gravatar for Ryan Thompson
6.5 years ago by
Ryan Thompson3.4k
TSRI, La Jolla, CA
Ryan Thompson3.4k wrote:

There are a couple of reasons. First, RNA is considered highly unstable, not because of any fundamental chemical instability, but because RNAses are ubiquitous in nature, so separating the RNA in a sample from all residual RNAses and then keeping that RNA sample free of RNAses can be quite difficult. In contrast, DNA is a lot more biologically stable, so converting to DNA ensures the stability of the sample's information content. (Edit: As pointed out in a comment, DNAses can easily be inactivated by chelating their metal ion cofactors, while RNAses do not require metal ion cofactors and are therefore much harder to inactivate.)

Second, PCR amplification only works on DNA, so unless you can obtain enough RNA to feed directly into your sequencing protocol, you need to amplify with PCR, and therefore you must reverse-transcribe to cDNA. In theory, you could probably adapt PCR to RNA using RNA-dependent RNA polymerase instead of DNA polymerase, but RdRp is a viral protein with a relatively high error rate (to facilitate rapid viral evolution), so an RNA-based PCR protocol would introduce far more errors than the combination of reverse-transcription and DNA-based PCR.

Third, most (all?) existing sequencing protocols are designed for sequencing DNA using DNA polymerase. Again, you could probably adapt the protocol to use reverse-transcriptase or RdRp in order to sequence directly from an RNA template, but again, RdRp is too error-prone, and if you're going to use reverse-transcriptase for the sequencing, you may as well use it initially to get cDNA and then sequence that. The overall error rate will be the same, and you will have DNA for most of the time instead of having to babysit your delicate RNA.

So basically, RTase has problems, but there's no alternative for sequencing or amplifying RNA that doesn't have even worse problems.

As for your second question: RTase takes a single-stranded RNA molecule as a template and synthesizes the complementary DNA strand, resulting in a duplex of one RNA chain and one DNA chain. Typically, after this process is complete, RNase H is used to degrade the original RNA template, leaving only single-stranded cDNA. This is then replicated using normal DNA polymaerase to form double-stranded DNA, which can then be amplified by PCR or processed in any number of other ways.

ADD COMMENTlink modified 6.5 years ago • written 6.5 years ago by Ryan Thompson3.4k
7

I'd just add to your great answer that it could be argued that RNA IS fundamentally chemically more unstable than DNA (particularly in basic environments) in that it is a lot more prone to hydrolysis because of the nucleophilic 2ยด hydroxyl groups. This hydroxyl group is also the reason that RNAses don't need metal ions for activity unlike DNAses. One of the reasons RNAses are harder to deal with because you can't just easily inactivate them via chelation with EDTA etc like you would with most (if not all) DNAses.

ADD REPLYlink modified 6.5 years ago • written 6.5 years ago by Sulfolobus140

Looks like you're right. "The problem, however, is that mRNA is very unstable outside of a cell; therefore, scientists use special enzymes to convert it to complementary DNA (cDNA)". Source: http://www.ncbi.nlm.nih.gov/About/primer/est.html

ADD REPLYlink written 6.5 years ago by Justin440

But if (when) large-scale single-molecule sequencing becomes viable, it might be possible to sequence RNA directly?

ADD REPLYlink written 6.5 years ago by Ketil3.9k
2

Single-molecule methods based on non-polymerase-based reactions might one day be usable to sequence RNA directly. With today's technology, if you could obtain a sufficient amount of RNA without needing PCR, you could maybe theoretically sequence it via SOLiD's sequencing-by-ligation technology if you could adapt the protocol to be RNA-friendly. In that case you would never involve any kind of polymerase or RTase.

ADD REPLYlink written 6.5 years ago by Ryan Thompson3.4k
0
gravatar for garethreid123
3.9 years ago by
Australia
garethreid1230 wrote:

Its a good question and still true. Lots gets lost in cDNA transcription and it is interesting to see the current Illumina TruSeq RNASeq assay still uses cDNA. I guess if they cant do it no one can. I think/hop this will change with PCR free protocols, soon.....

ADD COMMENTlink modified 3.9 years ago • written 3.9 years ago by garethreid1230
0
gravatar for SeqFreak
2.7 years ago by
SeqFreak0
SeqFreak0 wrote:

Looks like cDNA may not be a requirement in the future - direct RNA sequencing using nanopores preprint http://biorxiv.org/content/early/2016/08/12/068809

ADD COMMENTlink written 2.7 years ago by SeqFreak0

Yes very exciting technology and I'm eager to test it ;-)

ADD REPLYlink written 2.7 years ago by WouterDeCoster38k
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