It depends upon which organism we are talking about. If eucaryotes - then it is often observed with mitochondrial RNA and tRNA. Despite it is transcrybed from circular DNA and then cut into functional molecule, ratio of mature RNA content varies from tissue to tissue. Mainly it is linked to regulation of transcript stability, for example - changes in the length of polyA tail.

Thanks for the response Stepan, I should have specified that I'm curious for eukaryotes.

Hi Robert, there are many reasons for variable expression levels: alternative splicing, alternative start sites, alternative transcription rates, alternative polyadenylation, changes in transcript stability, etc. You would have to be more specific as to the situation you are referring to. Also, please keep in mind, that two PCR primers will have different efficiency in a real bench experiment, and there is no way you can quantitatively compare them directly. Hope it helps, best.

Thanks Olga, those are all great suggestions. This is a viral bicistronic gene that has a single promoter directing transcription of several spliced mRNA transcript variants for two proteins that can be translated (A and B): full A + full B, short A + full B, shorter A + full B. In other words, only the first of these spliced mRNA transcripts can be translated into functional protein A, whereas all 3 can be translated into functional protein B.

Now for assays: assay for A mRNA can detect all three transcripts while assay for B mRNA can also detect all three transcripts. Theoretically, there should be no difference due to the spliced variants because of this. Under some experimental conditions however, relative levels of A are higher/lower than B (not always as well). The other suggestions are quite interesting and I'll definitely be taking a closer look.

If you really want to compare the levels of different isoforms, I would suggested either radioactive RT-PCR or Northern Blot- these are old methods but they allow you to visualize and quantify different transcripts. If you are using RT-QPCR, you have to make sure that the primers are really specific to the isoforms, and to make sure you don't have any unexpected isoforms. Also make sure that the primers are above the exon-exon junction so you are sure to be looking at spliced vs non spliced transcripts. Good luck

Dear robert,

I understand you are talking about bacterial operons with different expression levels of individual genes. It has been described for individual operons and we have characterized that on a large-scale in Mycoplasma pneumoniae (Guell et al, Science 2009). We mainly find lower levels (staircases down) in downstream genes, but also other patterns. Using tiling arrays, we find that the steps occur particularly at the borders between genes indicating that there is some regulation of transcription elongation at these sites. However, we don't really know the mechanism for this. For the other cases, (steps up) there are internal promoters that can explain them. Hope this helps!

If you observe the slope of the amplification curve of the log-emission, you can judge the efficiency of the individual reactions, which you must have done if you dare to say that the reactions have equivalent efficiency.

In the first few cycles the original cDNA has a significant contribution as template. This molecule, being a single stranded nucleic acid, may have RNA-like structure, and if some of your primers should anneal to loci with structures, it can influence the chance of annealing/priming. This effect diminishes when the template is the already amplified short product. What you see on the amplificatioin curve is the amplification efficiency of this small molecule, which may be identical ideally, but if there is some delay in the amplification due to the template structure in the first cycles, that delay will be maintined throughout.

The above effect may or may not occur, but it should be constant under the same experimental conditions. Different treatments that alter splicing and therefore change the overall sequence, hence the possible structures, may also have some influence on the PCR efficiency in the first cycles in a treatment-dependent way.

In bacterial systems, where there is no splicing, but the multi-cistronic messages are common, we often encounter step-down pattern in the ORFs. This may as well be due to some in vivo 3 ' -> 5' exo-RNAse activity before we harvest the samples and freeze all enzyme activities. Another possible explanation is some random immature termination of the mRNA synthesis, which (if it realy happens) should naturally result in steadily decreasing amount of mRNA regions as we go from 5' to 3' direction.

Nevertheless, when we see a significantly more pronounced step-down between two ORFs in a -let's say- five-cystronic mRNA we can suspect some weak terminator structure there.

We sometimes see treatment-specific step-up in one point of the multicystronic operon. My strong guess is that there is a secondary (alternative, weak, funny)promoter there that may as well even overlap with the ORF upstream (a phenomenon that also happens frequently in such tightly packed genomes).

These are just some mere speculations, though.