Question: How much importance is RNAseq experimental bias?
gravatar for Jon
6 months ago by
United States
Jon130 wrote:

Hi there,

We did RNA sequence for 6 different treatments. Samples are from specific region of brain. But he took 5 mouse brains from one treatment and 7 mouse brains from other treatment (Reason: Ribotag IP gives 1 nanogram of RNA for 5-8 brains). Will that create big batch effect, Even if I normalize the data using normalizeBetweenArrays {limma} or something like this??

To be precise: Ribotag IP performed by pulling down ribosomes which expresses particular gene, then sequenced by SMART-Seq.

Have anyone came across this situation?


ADD COMMENTlink modified 6 months ago by Devon Ryan92k • written 6 months ago by Jon130

So you pooled 5 or 7 brains, pooled the RNA respectively and then prepared the libraries? Do you have replicates or is this then n=1 for every condition?

ADD REPLYlink written 6 months ago by ATpoint24k

yes, I have two replicates for very treatment.

ADD REPLYlink written 6 months ago by Jon130
gravatar for Devon Ryan
6 months ago by
Devon Ryan92k
Freiburg, Germany
Devon Ryan92k wrote:

The difference in the number of pooled brains isn't a particular problem, though hopefully those were pooled from litter mates and then litters used as biological samples. What you should watch out for is if some brains took longer to extract and process than others, since that will certainly lead to batch effects due to RNA degradation.

ADD COMMENTlink written 6 months ago by Devon Ryan92k

okay, but the amount of RNA from 5 brains will be different from 6 brains right?

When I look at the read counts/TPM matrix, column sums are higher in the sample pool of brains than 5 brains.

ADD REPLYlink written 5 months ago by Jon130

It will but as you input a fixed amount of RNA to the library prep it should not matter too much. For the uneven read number, this is normal as sequencing never produces 100% even read counts. This is not necessarily an issue based on RNA amount. This is where normalization comes into place.

ADD REPLYlink written 5 months ago by ATpoint24k
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