I was wondering if anyone had any input on what follows below:
I have a pretty straightforward experimental setup for de novo transcriptome assembly and differential expression analysis:
2 conditions: Stressed and unstressed
3 replicates for each condition
RNA extracted first by trizol/chloroform, followed by PolyA selection -> Illumina mRNA lib prep kit v2 (replicates individually indexed)
I get back ~half a billion reads. Pool everything and assembly a de novo transcriptome. I estimate abundance with RSEM for each of my 6 replicate samples and then run differential expression with edgeR.
What I find puzzles me (I am looking for genes that are upregulated in response to stress). Between my 2 conditions I get ~700 differentially expressed genes. I look at those upregulated in my stressed condition and BLAST results are ~99% unknown. I look at those genes down regulated in the stress condition and some of them all look like things that I would expect to be UP not down regulated.
Do you think I have completely messed up my experiment? Besides actual down regulation of a genes transcription I wonder what could explain these results.
If a transcript is translated a lot will that led to faster turn over?
If anyone has any ideas about some biological explanation, I love to hear it.
Cheers,
T
I have a pretty straightforward experimental setup for de novo transcriptome assembly and differential expression analysis:
2 conditions: Stressed and unstressed
3 replicates for each condition
RNA extracted first by trizol/chloroform, followed by PolyA selection -> Illumina mRNA lib prep kit v2 (replicates individually indexed)
I get back ~half a billion reads. Pool everything and assembly a de novo transcriptome. I estimate abundance with RSEM for each of my 6 replicate samples and then run differential expression with edgeR.
What I find puzzles me (I am looking for genes that are upregulated in response to stress). Between my 2 conditions I get ~700 differentially expressed genes. I look at those upregulated in my stressed condition and BLAST results are ~99% unknown. I look at those genes down regulated in the stress condition and some of them all look like things that I would expect to be UP not down regulated.
Do you think I have completely messed up my experiment? Besides actual down regulation of a genes transcription I wonder what could explain these results.
If a transcript is translated a lot will that led to faster turn over?
If anyone has any ideas about some biological explanation, I love to hear it.
Cheers,
T
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