I think the most direct assay for transcriptional activity is a run-on assay. Chip for Pol II is also used often for this purpose and I think its quite dependable. A really easy way to check this is to do qPCR spanning an exon-intron junction - pre-mRNA levels should correlate better to transcriptional levels than mRNA.

I've thought about this problem as well, since there are a growing number of papers that show a significant discordance between levels of transcript and protein, which calls the interpretation of may papers that rely solely on microarray, RT-qPCR and RNA-seq into question. It's necessary to validate microarray with qPCR but most people stop there, when they should be following up with ELISA, Westerns, etc. The new Applied Biosystems Vii7 qPCR machine has the ability to run protein expression assays through a proprietary kit, which means one machine, two complimentary assays.

If you do not want to do any protein work or use radioactivity in a run-on assay, another method that was used pre-qPCR exposed cell lines or embryos to a pol II inhibitor such as DRB, alpha amanitin, or Actinomycin D. In a properly designed experiment this should give you an idea of the amount of new/active transcript. Of course there are other downstream regulatory variables that this experiment can't address, but it's a good first step.

Try RNAseq, qRT-PCR, northern, western blots. If all conclude same results, the claim should very likely be correct.

My question addresses the notion that techniques such as microarray, RT-PCR, RNAseq and Northern blotting all evaluate transcript LEVELS, not transcriptional ACTIVITIES. This concern extends also to increased transcript half-life (or decreased decay), which would result also in an elevated protein level (independent of transcriptional activity).

Also the decision to ignore changes in transcript levels that do not effect protein levels is inherently flawed since several types of RNA (i.e. lincRNA and miRNA) are functional (and biologically relevant) without the generation of any proteins. Also it is important to remember that proteins are frequently translated in a non-functional form - either from subcellular localization or a need for post-translational activation (cleavage or covalent modification, like phosphorylation).

This however takes the discussion from the original question into a question of biological relevance, which albeit important is an entirely different discussion.

"since All these transcripts that are being formed, should logically have some biological function, as a cell wouldnt expediate precious energy in transcribing mrnas which then do not form proteins or mrnas which do not have any biological function"

The cost of transcribing is probably not that much since most of the mammalian genome is pervasively transcribed, leading to lots of non-conserved spurious transcript.

Here's a paper that's germaine to the discussion

"Comparing Transcription Rate and mRNA Abundance as Parameters for Biochemical Pathway and Network Analysis"

with pulse chase radioactive labelling experiments you can deduct the half life of your rna. If that does not change and the steady state expression increase, then is transcribed more than before.

-same half life

-increased steady state levels

then upregulated at the transcriptional level

Eventually a reporter assay can confirm that

Hi,

In line with what Margarida mentioned above, run-on would be the way to go. Either using radioactivity or with labelled nucleotides. If you're more interested in global changes, GROseq, NET-seq and related methods might be interesting for you (which really measure active transcription in contrast to mere RNAseq).

Although any of these techniques may help you establish a link between the rate of transcription and mature transcript levels of your gene in one or more specific conditions, I don't think you could claim that this relation will hold if you alter the conditions. As you mention yourself, different conditions will likely also affect a number of co-transcriptional and post-transcriptional regulatory mechanisms which could significantly change the relationship between transcription rate and transcript levels.

I guess if you want to know your transcription rate, you have to measure it directly.