I have been out of the field for many years now. You may want to ask your question of Sam Benchimol out of York University, Toronto ([email protected])

please can you explain how you do your experiments? do you stress cells (DNA damage, heat, ...)? sometimes we detect also pre-spliced mRNA. have you defined the basal level of splicing? (meaning do you have more frequently "intronic sequence" in p53 mRNA than in Actin mRNa?) you can exchange by email if you want)

thanks jean-christophe

our initial experiments are with a panel of cell lines that have not been stimulated or stressed - however, appears much above the basal level of intronic retention as these RNA species (with various introns) are very frequent

in what circumstances have you seen intronic p53 retention?

Dose your PCR primer spanning the splicing junction? If not, the "intronic" sequences may be DNA contamination.

yes- the PCR primers span multiple exon junctions

Very interesting. Is it specific to some kind of conditions or cells? Normally using oligo dT or random hexamers you will get some level of unspliced RNAs. You are probably doing total RNA preparation and what you are seeing is just that, unspliced RNA or rather nascent RNA molecules. You might also have just a few introns "retained" , typical of some introns which probably are spliced later that any other in the same transcript. Why that happens I do not know, we see more of an intron signal on exons which are alternative spliced or that require some other kind of processing. If you do not want the intronic sequences in your RT-PCRs I should recommend you to do a CYTOPLASMIC RNA prep, there are commercial kits available.

It very much depends on how many of these unspliced RNAs you find as compared to the processed mRNA. If you look at sufficient depths you will find almost any possible form of the RNA yet normally a few splice variants including the one considered "normal" or "full length" outweigh the unspliced forms by far.

we always detect intronic retention because of pre-mRNa splcing but I mean here that we keep several full introns. We never detect only part of intron (we detect part of intron when it is genuine splice variant like p53beta or p53gamma (cryptic exon in intron9 of human p53).

do you detect full introns or only part of introns?

to role out possible problem of genomic DNA contamination despite using DNase in our extraction methods, we do a PCR using total RNA extract that have not been reverse transcribed. if you amplify something it means that you have DNA in your RNa prep.

THe other problem is that oligodT and hexamer primer (random primer) can somehow stabilise RNa loops Structure, which would lead to artefactual splice variant. To avoid that we perform the reverse trasncription at 45C. please download my articles on how to detect and quantify p53isoforms mRNA and protein. we can download them freely.

Regulated intron retention is a known post-transcriptional regulation mechanism; it typically results in degradation of the message prior to translation. The etiology of such regulation is usually through splicing enhancers or silencers, which typically bind RNA sites close to the splice site(s) they regulate either in the exon or the intron.

However, it's important to demonstrate that you're looking at _regulated_ intron retention, rather than simply a slow splicing reaction that just hasn't happened yet. You may therefore want to look at the _order_ in which the introns in your gene are typically removed. If you can show that your retained introns are most often removed before other introns in the same transcript, you can claim that the intron is being retained in a regulated manner.

Ulrike Schwartz and Peter Byers have developed some good methodology for determining the order in which introns are spliced; they've worked in this area for at least the past decade or two.

PCR is extremelly sensitive

a number of premessenger mRNA may not yet be spliced, and you would see them

It is frequent in RNAseq to observe that some introns are spliced slower than others