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RNA seq and Invitro data not matching

Hi all, Invitro data shows upregulation of certain genes where as RNA seq analysis shows a positive foldchange for same genes but not significant p value and q value. How can i justify this?

rna-seq

What is Invitro? qPCR? Please add details: How many replicates, what is the setup, does PCA indicate good reproducability between replicates? What is the tool you used for analysis? Also please show an MA-plot and highlight the gene you are interested in. Your experiment could simply be underpowered.

More info needed. How was RNAseq performed and analysed, what's invitro data and how was it analysed.

1 answer

All experimental technique have a limit to their precision. Thus, the estimated foldchange we get out of both qPCR and RNAseq is just that - an estimate. That is why we put error bars on things - the error bars (at least if they represent the 95% confidence interval, which they normally should) more or less tell us the range of values that could plausibly be true. In RNAseq, these error-bars can be really quite wide if there is a small number of replicates or for a lowly expressed gene. The fact that this gene is not significant in RNA-seq analysis means that the error-bars cross 0. That is both negative and positive logfold changes could plausibly be true. This is what it means for a gene to not be significant. If you did yuor analysis in DESeq2, then the standard error is in the output table and you can calcaulte the 95% confidence interval for the fold change. Here is an extract from the DESeq2 vignette:

    ##                     baseMean      log2FoldChange             lfcSE
    ##                    <numeric>           <numeric>         <numeric>
    ## FBgn0000008 95.1442917575889 0.0227644122547027  0.22372865161848
    ## FBgn0000014 1.05652281859341  -0.495120386253493  2.14318579304427
    ## FBgn0000017 4352.55356876647   -0.23991894353759 0.126336905404352

For FBgn0000008 the best guess log2FoldChange is 0.022, but the standard error is 0.223. The 95% CI is very approximately 2SE, so the range of log2FoldChanges that could feasibly be true is 0.022 -/+ 20.22 = 0.022 +/- 0.44 or -0.42 to +0.46. Of course, all this also applies to results from qPCR.

I changed this answer because I miss-read the question and thought the "invitro" data and the RNAseq were going in opposite directions.

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