total RNA-seq vs mRNA-seq

[gene_x]

I'm new to RNA-seq and I've been reading about it. I'm wondering what's the disadvantage of doing total RNA-seq compared to mRNA-seq if money is not an issue. It seems to me that total RNA-seq would gave more data about other RNA species than mRNA so it appears to be a better method. Or does total RNA-seq suffers on mRNA quantification compared to mRNA-seq so if you are only interested in mRNA, then mRNA-seq is the way to go?

If you could share your experience or thoughts on this or point to some reading material, it would be greatly appreciated!

[mikep]

It can also depend on your sample. If you have miniscule starting material and need some serious amplification then the protocol may require mRNA as a starting point. In addition if you are going to do totalRNA you should be performing a stranded protocol as well, which is less of an issue for mRNA.

ETA: This isn't really a bioinformatics issue.

[gene_x]

It can also depend on your sample. If you have miniscule starting material and need some serious amplification then the protocol may require mRNA as a starting point. In addition if you are going to do totalRNA you should be performing a stranded protocol as well, which is less of an issue for mRNA.

ETA: This isn't really a bioinformatics issue.

Thanks for your reply!

Why would having miniscule starting material may require mRNA as a starting point? Does that suggest you can't do total RNA seq if you have minuscule RNA? I don't understand it..

Also why would stranded protocol be less of an issue for mRNA than total RNA? I thought stranded protocols have no difference in total RNA-seq vs mRNA-seq?

PS: I haven't really posted in other sections yet.. will be more specific in the future.

[mikep]

Thanks for your reply!

Why would having miniscule starting material may require mRNA as a starting point? Does that suggest you can't do total RNA seq if you have minuscule RNA? I don't understand it..

If you have a small initial sample you want it as enriched as possible for the stuff you actually care about. For most purposes, the non mRNA you get from totalRNA is a nice to have, not a must have.

Also why would stranded protocol be less of an issue for mRNA than total RNA? I thought stranded protocols have no difference in total RNA-seq vs mRNA-seq?

~50% of the reads from a totalRNA sample will map to things not mRNA (at least in my hands). In addition, since alot of the lncRNA overlap mRNA, you need the strandednesss to tell which transcript the reads actually belong to.

If you are pulling down mRNA then strandedness is less of an issue.

[gene_x]

If you have a small initial sample you want it as enriched as possible for the stuff you actually care about. For most purposes, the non mRNA you get from totalRNA is a nice to have, not a must have.



~50% of the reads from a totalRNA sample will map to things not mRNA (at least in my hands). In addition, since alot of the lncRNA overlap mRNA, you need the strandednesss to tell which transcript the reads actually belong to.

If you are pulling down mRNA then strandedness is less of an issue.

I see. Those make sense.

It's a bit surprising to know that 50% of the total RNA reads map don't map to mRNA...

[kentawan]

I see. Those make sense.

It's a bit surprising to know that 50% of the total RNA reads map don't map to mRNA...

Those may be interesting things like miRNA/lncRNA. If you have the resources you should look into them as well.

[gene_x]

Those may be interesting things like miRNA/lncRNA. If you have the resources you should look into them as well.

Yeah, sure. Make the best use of your data.

[pmiguel]

gene_x,
It is important to distinguish RNAseq from "total RNA" vs from "rRNA-depleted total RNA". "Total RNA" to a bench scientist, means the RNA present in a raw RNA prep, with no depletion of any kind done. Typically total RNA will be >90% ribosomal RNA.

It is very rare to sequence total RNA. People almost always do some sort of depletion. The most common type of depletion is poly A+ binding. By washing away RNA species without a poly A tail, you rid yourself of most of the rRNA.

As noted, you also lose many other species of RNA that lack the poly A tail. But, unless you have a special interest in these, you are better off removing them anyway. They just dilute out information most commonly sought by these sorts of experiments.

Also, in many cases, the functions of many types of non-polyA, non-rRNA are poorly understood. So you are often going into "de novo" territory by including them in your data set.

--
Phillip

[gene_x]

gene_x,
It is important to distinguish RNAseq from "total RNA" vs from "rRNA-depleted total RNA". "Total RNA" to a bench scientist, means the RNA present in a raw RNA prep, with no depletion of any kind done. Typically total RNA will be >90% ribosomal RNA.

It is very rare to sequence total RNA. People almost always do some sort of depletion. The most common type of depletion is poly A+ binding. By washing away RNA species without a poly A tail, you rid yourself of most of the rRNA.

As noted, you also lose many other species of RNA that lack the poly A tail. But, unless you have a special interest in these, you are better off removing them anyway. They just dilute out information most commonly sought by these sorts of experiments.

Also, in many cases, the functions of many types of non-polyA, non-rRNA are poorly understood. So you are often going into "de novo" territory by including them in your data set.

--
Phillip

Thanks for the reminder. Yeah, I do understand that even for total RNA sequencing, they would get rid of rRNA first and the difference between mRNA-seq and total RNA-seq is mostly due to how you get ride of rRNA.