Question: Is mixing samples (tissues from several mice in one replicate) OK for RNA-seq? and does it reduce the requirement for replicates?
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gravatar for NGS_enthusiast
3 months ago by
NGS_enthusiast0 wrote:

Hi, I was wondering if mixing samples (we probably have to, in order to have enough RNA) was OK? I was also wondering if it could increase reliability from few replicates, since lots of samples mixed in one replicate would help reduce variability? For example maybe 2 replicates of 5 mice each would be better than 4 replicates, each from 1 mouse. thanks a lot for your input, best regards

rna-seq • 228 views
ADD COMMENTlink modified 3 months ago by swbarnes29.4k • written 3 months ago by NGS_enthusiast0
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How much RNA are you getting out per animal? It is simple, if you don't get enough RNA for a per-mouse prep then yes mix it, since there is no alternative. It is not uncommon to do that, specially for assays like ChIP-seq where you need many cells or if material is super rare. There are today low-input RNA-seq kits though. You can use them with a few hundred or thousand cells, required quite some PCR amplification though.

ADD REPLYlink modified 3 months ago • written 3 months ago by ATpoint44k

yes thanks well I was pondering whether to try low input or keep high input and mix samples.

ADD REPLYlink written 10 weeks ago by NGS_enthusiast0

And what did you decide for eventually?

ADD REPLYlink written 10 weeks ago by ATpoint44k

For this experiment, I decided to mix some samples as low input libraries are not yet available at the facility where we do our sequencing. But I'll try to change in the future.

ADD REPLYlink written 10 weeks ago by NGS_enthusiast0
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gravatar for swbarnes2
3 months ago by
swbarnes29.4k
United States
swbarnes29.4k wrote:

For example maybe 2 replicates of 5 mice each would be better than 4 replicates, each from 1 mouse.

No. Most of the point of replicates is to understand how much the gene naturally varies among the replicates; averaging them all together destroys that. 4 separate replicates is absolutely better than smooshing 2 groups of 5 mice into two combined samples.

ADD COMMENTlink modified 3 months ago • written 3 months ago by swbarnes29.4k
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