I ran an RNAseq with Brugia malayi worms treated with a drug and untreated. I found a certain number of genes differentially expressed in the treated worms as compared to the untreated. I selected one upregulated gene to conduct confirmatory qPCR. I have selected Actin to be my control for the study as its expression is unaltered. Should I consider the spliced version of the gene to build the primers or unspliced version which has the intron? The aim is to not amplify the background genomic DNA. Many literature articles say use exon-exon span to design primers. What does this mean exactly?
If you use a tool like "NCBI primer blast" to design your primers, you will have the oportunity to request that your amplifyed sequence starts in one exon and ends in the following (exon exon spab). As an intron can be very long, there is no chance to amplify an intron in a classic qPCR (30'' extension, e.g.). Then, if there is any genomic DNA contaminating your sample, this will not be amplifyed and your quantification will be more reliable.
If you use a tool like "NCBI primer blast" to design your primers, you will have the oportunity to request that your amplifyed sequence starts in one exon and ends in the following (exon exon spab). As an intron can be very long, there is no chance to amplify an intron in a classic qPCR (30'' extension, e.g.). Then, if there is any genomic DNA contaminating your sample, this will not be amplifyed and your quantification will be more reliable.
Hi Amruta,
The designing of primers in exon-exon junction basically means that you design a part of the primer in one exon and the the remaining part in the adjacent region from the next exon (the regions will be continous in the mature transcript)..One should work in the mature trasncript and not in the unspliced transcript...the levels of mature transcript are likely to correlate more to the observed phenomenon...(or final protein)..bye
You will want to use the processed mRNA (spliced) sequence unless you want to specifically measure unprocessed transcriptional response. By creating a primer set that spans an intron, you will only amplify processed mRNA since this sequence is unique when the introns present in the template DNA and unprocessed mRNA are spliced out.
As mentioned above (Rodrigo Aguilar, Ajay Saini) your best choise would be intron-spanning primers to distinguish from gDNA
Human actin has very short introns (like 50-100 bases). In that case, intron-spanning primers are not very efficient in distinguishing cDNA from gDNA. If that's the case for your organism too, use primers on exon junctions if it is possible to design decent primers that way.
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