Hi everyone, im interested into knowing the quantity of spliced and unspliced mRNA for a paticular gene. I thought using primers for mature mRNA (exon to exon) and other set of primers to quantify primary transcript (pre-mRNA) using a part of intron and its contiguous exon to amplify unspliced mRNA. Is this procedure correct? Can it be donde by RT-qPCR if i treat my extracted RNA with DNAsa (so theres no genomic contamination)?
Thanks
That sounds possible. You will need a control for DNA contamination (despite using DNAse!), this chould be primers agains some genomic sequence.
If you are interested only in relative changes between pre- and mature RNA (under different conditions), you might set up a multiplex PCR using on forward and two reverse primers. One probe could target the second exon, the other could target the intron. This way you can get the relative amounts in a single PCR. These results (dCt values) may then be compared between conditions. However, the multiplex assay may not work well when the relative amounts of pre- and matrue RNA are drastically different.
If you are interestend in real concentrations (or ratios thereof) then you need external standards for both amplicons and in this case I would go for singleplex reactions.
Not with the standard cDNA synthesis protocol. It relies on the dTTT primer to bind and amplify mRNA to cDNA based on the mature mRNA with a polyA tail.
Introns are removed and exons are spliced together BEFORE the polyA tail is added.
But there are protocols for you. Do a literature search.
Good luck!
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