Question: why dont we need technical replicate in case of RNA Seq.?
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gravatar for neeraj69_sbt
2.4 years ago by
neeraj69_sbt0 wrote:

I want to do RNA seq comparison between two situation in case of plant. is it necessary to use technical replicates in this case. RNA seq is new emerging technology to detect allelic isoform expression as well as DGE in different situation.?

rna-seq • 1.1k views
ADD COMMENTlink modified 2.4 years ago by WouterDeCoster40k • written 2.4 years ago by neeraj69_sbt0
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I would like to link my answer to Are different colonies from the same cell line valid biological replicates for DESeq 2? about the distinction (or lack thereof) between what are "technical" and "biological" replicates.

ADD REPLYlink written 2.4 years ago by dariober10k

You'll need replicates if you want to publish your results.

ADD REPLYlink written 2.4 years ago by russhh4.7k
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Important to differentiate between biological and technical replicates.

ADD REPLYlink written 2.4 years ago by WouterDeCoster40k
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gravatar for WouterDeCoster
2.4 years ago by
Belgium
WouterDeCoster40k wrote:

Since the technical reproducibility of RNA-seq is rather good technical replicates are not really necessary. This means that if one skilled person uses the same library prep kit on 5 aliquots of the same sample and creates 5 libraries, and sequences on the same sequencer, the result should be 5 times the same data, approximately. There will be some "Poisson variability", but technical variability is limited.

(Note that if a different kit is used results will be different, obviously.)

But the difference between two plants (of the same species) will be bigger, because of biological variability. So in order to accurately quantify a difference between two groups, you need to assess the intra-group variability, the biological variability. To properly characterize the biological variability you need biological replicates.

The very minimum of replicates would be 3 samples per group. But if you can afford it, definitely do more samples.

ADD COMMENTlink written 2.4 years ago by WouterDeCoster40k
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There's a school of thought currently that even 3 is no where near enough to get the power that RNAseq needs (https://www.ncbi.nlm.nih.gov/pubmed/27022035 ). 12 replicates would be crazy expensive however, so everyone I know does just do 3.

ADD REPLYlink modified 2.4 years ago • written 2.4 years ago by Joe14k

Hmmm valid remark and interesting reference. I would have assumed this also depends on how big this biological variability is. If you have inbred littermate mice you probably need less than having very different human subjects. However, in the paper you linked they work on (I assume well-controlled) yeast samples.

ADD REPLYlink written 2.4 years ago by WouterDeCoster40k

Yeah I can imagine that's fairly reasonably to assume. We work on (in theory) clonal bacterial populations in my lab, but its surprising how varied even that can be (as many of my lab mates, to their annoyance, would attest!).

ADD REPLYlink written 2.4 years ago by Joe14k
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gravatar for geek_y
2.4 years ago by
geek_y9.8k
Barcelona
geek_y9.8k wrote:

I don't think "RNA seq is new emerging technology". You don't need technical replicates but you need biological replicates. For allele isoform expression, you need many samples ( as you need to work with genotypes ) with more sequencing depth than that is required for DGE.

ADD COMMENTlink modified 2.4 years ago • written 2.4 years ago by geek_y9.8k
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