Technical/Biological Replicates In Rna-Seq For Two Cell Lines
Entering edit mode
10.5 years ago
Bio_X2Y ★ 3.9k

I have a question around the meaning of "biological replicate" in the context of applying RNA-seq to compare two cell lines. Apologies if this is an overly naeve question.

We have two human cell lines, one of which was derived from the other. Both have different phenotypes, and we want to use RNA-seq to explore the genetic underpinnings of the difference.

If we generate one cDNA library for each sample, and sequence each library on two lanes of an Illumina GA flowcell, I understand we will have "technical replicates". In this scenario, we can expect very little difference between the two replicates in a sample. If we were to use something like DESeq to call differential expression, it would be inappropriate to treat our technical replicates as replicates in DESeq, since that would likely lead to a large list of DE calls that don't reflect biological differences.

So, I'd like to know if it possible within our model to have "biological replicates" with which we can use DESeq to call biologically meaningful differential expression.

So, two questions:

(1) If we grow up two sets of cells from each of our two cell lines, generate separate cDNA libraries (4 in total), and sequence them on separate lanes, would these be considered "biological replicates" in the sense that it would be appropriate to treat them as replicates within something like DESeq. I suspect not, since the fact that both replicates in a sample derive from a single cell line within a short period of time will mean that they will be very similar anyway, almost as similar as the technical replicate scenario. Perhaps we would need entirely separate cell lines to be considered biological replicates.

(2) In general, how would others address this - does it seem a better approach to go with separate cells and separate libraries, or would this entail extra effort for effectively no benefit?

Thanks for your time.

replicates illumina rna • 14k views
Entering edit mode
10.5 years ago

Two "biological replicate" are two samples that should be identical (as much as you can/want control) but are biologically separated (different cells, different organisms, different populations, colonies...)

You want to check the difference between cell Line A and cell line B. Let's start assuming they are identical. Even if they are, by random fluctuation, technical issues, intrinsic slightly different environments... you will never observe that all genes have exactly the same expression. You find differences but can't conclude if they are inevitable fluctuation or result of an actual difference.

So, you want to have 2 independent populations from A and two independent populations from B and then see how the variability WITHIN A1 and A2 compare to B1 and B2. The RNA levels from A1 and A2 WILL NOT be the same because... because biological system are far from being deterministic. They might be very similar, but different.

because A and B would be on different plates (their environment) I would seed A1 and A2, B1 and B2 the same day on 4 distinct (but as similar as possible) dishes, grow them together in the same condition to minimize external influence, and then collect at once from the 4 cell lines, extract RNA...

Since the cost is not growing cell lines, but sequencing, I would recommend to do 4 independent replicates for A and for B (or any other cell lines you may be interested in) in ONE GO, and then freeze the sample or the RNA. Even better, if you could have somebody to give you the lines called alfa, bravo, charlie, delta... (make sure they keep track of what they are in a safe place ;) ) so that you are not biased while seeding, growing and manipulating the lines, that would be even better!

Entering edit mode
10.5 years ago

"Biological replicates" means different things under different experimental questions, but I think most observers would feel that if you split a colony, grow the two dishes to confluence, and separately measure a phenotype on the two populations, what you're measuring is the variation in your in vitro conditions and not a "biological" difference. If there were some defined treatment that you were testing while the cells grow-- response to treatment X that is expected to change phenotype in some interesting way-- that then I think you would have a better case. In my opinion, you'd consider two cell lines derived from separate humans (or tumors, or whatever) to be biological replicates.

Let's say you're looking at human tumor cell lines A1 and A2, where A2 is derived from A1 but has undergone epithelial-mesenchymal transition. I think you'd get the most benefit from measuring A1 vs. A2 in one run and then identifying promising candidate differences to test in a low-throughput manner (allele-specific taqman, etc) on lines B, C, D, and E that have similar phenotypic properties.


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