Question: RNA-SEQ: Which pairwise comparison for cells applied with low and high levels of mechanical forces?
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gravatar for Muha0216
20 months ago by
Muha02160
Muha02160 wrote:

hi guys,

i am a total noob when it comes to RNA-seq and nobody in my lab has done this technique before since we are generally a protein lab.

so basically i have gotten back rna-seq data in which theres 3 groups: a) uncompressed cells (control), b) cells compressed at low pressure, c) cells compressed at high pressure. Each group we sent RNA in 3 biological replicates. DEGs filter is set at >2 fold change, with p value <0.05

-The comparison between control vs cells compressed at low pressure yielded about 350 upregulated genes, 70 downregulated genes.

-Comparison between control vs cells compressed at high pressure yielded 58 upregulated genes, 15 downregulated genes (WEIRD??)

-Comparison between cells compressed at low pressure vs cells compressed at high pressure yielded 155 upregulated genes, 390 downregulated genes.

Issue 1: How in the world do i explain why there is such a lower number of DEGs obtained for control vs cells compressed at higher pressure? Can anyone here suggest some reasons? There is no problem in the quality of RNA-seq technique/methodology. The QC that the company ran on the RNA quality and reads is satisfactory (passed).

Issue 2: How would you present the data? From what i see, control vs cells compressed at low pressure the data seems promising. But i just feel like hiding the fact that i did control vs cells compressed at high pressure since i got such a low number of DEGs. Should i analyse common and unique DEGs from control vs cells compressed at low pressure.and control vs cells compressed at high pressure -which i dont think they will be many common DEGs

..... OR should i directly compare cells compressed at low pressure vs cells compressed at high pressure??

will appreciate any advise on this matter! many thanks!

sequencing rna-seq • 577 views
ADD COMMENTlink modified 20 months ago • written 20 months ago by Muha02160
1

You have described the technique you used in this experiment but have not said anything about what is the pressure supposed to do? Are you testing a hypothesis that cells are sensing pressure or are you checking if they are resistant to physical disruptive forces? It would help to add some background.

There is no set number of genes one should expect as DE. You have an adequate number of DE genes. DE genes (as many as possible within budget/effort) need to be verified (using an independent technique) that they are indeed responsible for whatever you are looking for.

ADD REPLYlink modified 20 months ago • written 20 months ago by genomax73k

Hi genomax,

there is no specific hypothesis. We are looking at the effects of varying levels of compressive pressure on the global gene expression of cancer cells. An expanding tumor in vivo experiences some degree of compressive pressure, and this pressure increases the bigger the tumor is. So we wanted to study and predict how such compression can affect biology of tumor. Genes that respond to this compressive pressure are termed as 'mechanosensitive'.. and we are hoping to see some oncogenes/tumor suppressors that are differentially regulated. Problem is i see a much much lower number of DEGs when i expose cells to higher pressure.

any suggestions on whether i should compare control vs low pressure & control vs high pressure or

low pressured cells vs high pressured cells?

much appreciated!

ADD REPLYlink written 20 months ago by Muha02160
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