Hi,

I would like to check the definition of the data that INSPEcT labels as the 'total' RNA, as output by this function: ratesFirstGuess(nascentInspObj, 'total'). In the INSPEcT papers, generally the two populations of RNA are referenced as premRNA (P) and mature mRNA (M). Is the 'total' RNA level output by this function indeed total (= P + M) or is it mature (M)? I have some analysis I would like to do with the mature RNA. If 'total' = 'mature' + 'preMRNA', then could I infer the 'mature' RNA level by simply subtracting, i.e. 'mature' = 'total' - 'preMRNA' using the 'total' and 'preMRNA' quantification from INSPEcT?

A second related question: What are the units of the 'total' and 'preMRNA' values output by: ratesFirstGuess(nascentInspObj, 'total') and ratesFirstGuess(nascentInspObj, 'preMRNA')? RPKM?

Thanks,
MK

Hi,

The total RNA (T) obtained by "ratesFirstGuess" and "viewModelRates" methods is the sum of the mature (M) and premature (P) RNAs, i.e. T = M + P. In case you are interested in obtaining mature RNA concentrations, you must subtract the output of premature concentrations from the total ones, either with

matureRNA <- ratesFirstGuess(nascentInspObj, 'total') - ratesFirstGuess(nascentInspObj, 'preMRNA')

or:

matureRNA <- viewModelRates(nascentInspObj, 'total') - viewModelRates(nascentInspObj, 'preMRNA')

according to the level of modeling you are interested in. Instead, in case you have a time-course design and you are interested in visualizing the mature RNA response, you can just set the argument "plot_matureRNA" to TRUE within the method "inHeatmap":

lfc <- inHeatmap(nascentInspObj, plot_matureRNA=TRUE)

The method "inHeatmap" also returns the log2 ratios of the concentrations and rates relative to time zero. Therefore, in case "plot_matureRNA" is set to TRUE, log2 ratios of the mature RNA are returned.

Regarding your second question, the units of Total, Mature and Premature RNAs depend upon the input you provided to INSPEcT. If you provided BAM, BigWig files, or read counts, INSPEcT calculates RPKMs out of them (methods "quantifyExpressionsFromBAMs", "quantifyExpressionsFromBWs", or "quantifyExpressionsFromTrCounts"). In case you started your analysis from transcripts abundances (via the method "quantifyExpressionsFromTrAbundance"), the unit is the one provided by the uses, that might be diverse (RPKM, TPM, molecule numbers, concentrations, etc.)

Best,

Stefano