Difference of 5' and 3' mRNA analysis
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2.9 years ago

Hi all,

I wanted to ask if someone can explain to me the differences of analyzing transcriptome profile using the 3' end information and the 5' end information.

Our lab recently tried using the 10x Genomics single cell technology and we have 2 types of libraries prepared for 8 samples for 2 different conditions (healthy and infected). 4 libraries (2 healthy, 2 infected) are prepared using the 3' Gene Expression kit while another 4 (2 healthy, 2 infected) are prepared using the 5' Immune Profiling kit.

Analysis was done using Seurat and in the end, I tried to look into the differential expressed genes between the 2 conditions and found that the DE genes are totally different between the 2 kits used. For example, gene A is found to be upregulated in healthy condition using the 3' kit but was upregulated in infected condition instead from the 5' kit data.

So I wanted to understand is what is/are the difference(s) between using the 5' end and 3'end of mRNA for transcriptome analysis and gene expression profiling. What exactly is the kind of information that we can obtain from using the different kits?

What I know is that using the 3' ends of mRNA is something like the gold standard for gene expression profiling. While for the 5' ends, the information we can get is more of start of transcription. Probably that's the reason why I got different results?

Could someone please advice? Thank you very much.

RNA-Seq transcriptomics 10x • 2.2k views

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