Hello Sue,

another possibility is that authors suspected intron retention, which could be due to either the presence of weak splicing signals or to the presence of splicing variants, as reported in many tumors. In this scenario the best option would be to perform oligo-dT purification in order to get rid of immature RNA (in this case you want to check the presence of your intron AFTER the splicing) and proceed with quantification. A caveat which is valid for all these situations is that you could co-amplify contaminating genomic DNA, therefore it's always advisable to use non retrotranscribed RNA as a negative control template in your Q-PCR assay. In these control wells you expect minimal to no signal. If signal is comparable with retrotranscribed wells, it means that gDNA contamination occurred and that you should treat your RNA with DNAse before Q-PCR.

Hi, Sue

It would be easier to comment if you could share the links for these papers.

Probably your question refers to the fact that in the most common protocols cDNA are synthesized using oligoDT. However, it is possible to synthesize cDNA using random primers. In any case, both random primers and oligoDT (which theoretically duplicates only mRNA single strands) may start cDNA synthesis from nuclear (also known as immature) mRNA, which contains introns.

best regards, 

Hi

When you extract total RNA from eukaryotic cells, your extraction yield contains all types of RNA in the cell. Immature mRNA is one of them which contains both exon and intron sequences. If you want to amplify or quantificate these immature types which are an indicative of expression level of introns, you have two choices:

1- After total RNA extraction for real-time PCR you can use random primers as alexandre has said. But to make sure that the amplified sequences are immature types, you should design your probe exactly on first exon-intron junction. This action ensures that the amplified sequences contain intron sequences. This method is not recommended for qPCR.

2- The second choice which is recommended and can be applied for qPCR, is to design your primers (or both primers and probe) exactly on exon-intron junctions.

Hello Sue,

another possibility is that authors suspected intron retention, which could be due to either the presence of weak splicing signals or to the presence of splicing variants, as reported in many tumors. In this scenario the best option would be to perform oligo-dT purification in order to get rid of immature RNA (in this case you want to check the presence of your intron AFTER the splicing) and proceed with quantification. A caveat which is valid for all these situations is that you could co-amplify contaminating genomic DNA, therefore it's always advisable to use non retrotranscribed RNA as a negative control template in your Q-PCR assay. In these control wells you expect minimal to no signal. If signal is comparable with retrotranscribed wells, it means that gDNA contamination occurred and that you should treat your RNA with DNAse before Q-PCR.

 In general, introns are transcribed but excised before the translation so mRNA has no introns.. DNAse treatment of extracted RNA samples before the RT step is recommended to avoid any gDNA but has certain disadvantages like the non-specificity issues of DNAse (except u have the Shrimp Nuclease, which is somewhat specific but we all can't have it) and also by law any type of gDNA removal protocol get results in loss of RNA quality and quantity which is quite lethal for my qPCR..and we don't want it at all, so likely we have some gDNA always in our samples which we know has introns and exons both not like the cDNA having pure exons. And we all of course don't want this gDNA amplification for our qPCR. So qPCR primers are designed in a way that they can skip the introns (from gDNA). That is why extracted RNA samples also run  as controls in qPCR reaction (this control well has everything of the reaction except the cDNA template made by that particular RNA) to see there is any gDNA in my samples or not........

 Thank your for all the answers. 

According to Andrey S Bavykin's advice, I think I will ask my question in another way. 

This is the paper I refer to, which they did real time PCR to check intron expression level with cDNA as template in Figure 5. They did transcription elongation recovery assay and test expression levels by qPCR in different genomic regions, which includes introns. 

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0126654

Actually, I plan to do the similar experiments, but I am just wondering whether their method is reliable. 

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0126654