Dear Mohamed

exon junction or intron spanning primers are definitely the best as they target specifically RNA and not parent genomic DNA. Even if the genomic DNA is much larger is size and therefore will not realistically amplify under the time limits of PCR designed to amplify size limited qPCR products (100-300bp) nevertheless is can drain your principal qPCR reaction and under mine priming efficiency

Thus, exon spanning primers are best. In that regard primer BLAST and 2 sites by IDT (primer quest) and Roche (universal qPCR primer design tool) are excellent in my experience:

1. http://www.ncbi.nlm.nih.gov/tools/primer-blast/

2. http://www.idtdna.com/primerquest/home/index

3. https://lifescience.roche.com/webapp/wcs/stores/servlet/CategoryDisplay?tab=Assay+Design+Center&identifier=Universal+Probe+Library&langId=-1

In all 3 cases you will need to find your NCBI ref sequence (from NCBI Gen Bank) and for the IDT and Roche tools input the ref sequence as a FASTA file or simply type the Ref Seq accession # and you will get back exon spanning (qPCR) primers embedded in your ref sequence

Primer BLAST incidentally is particularly effective for making spliceform specific primers whereas Primer quest and universal primer design tolls are more direct

Sometimes, in my experience, exon spanning primers do not amplify as efficiently as internal exon primers. Thus you can perform qPCR with internal exon primers but in this instance make sure you have rigorously removed genomic DNA ny DNAse I digestion

Qiagens RNAse free DNAse I and PE (Ambion) Turbo rDNAse are particularly effective in that regard

1. https://www.qiagen.com/gb/shop/lab-basics/enzymes/RNase-Free-DNase-Set?cmpid=QVen2GADNase&kwid=%2bqiagen+%2brnase+%2bfree+%2bdnase#orderinginformation

2. https://www.thermofisher.com/order/catalog/product/AM2238

The latter in particular allows you to aggressively digest RNA for 1 hour at 37C without concomitant digestion of parent RNA, a problem with removing GDNA from total RNA when digesting @ 37C for > 30 min (with many DNAse I preps)

If you do decide to go down the exon spanning qPCR primer route - rather than internal exon primers + DNASe I digestion route (although the latter will work particularly for genes with large introns) find attached 2 x SOPs detailing how to use IDT primer quest and also primer BLAST 

Hi,

 primers don't need to span a exon - exon junction but they should be separated by a intron. 

 Also it depends on the likely hood the off target being produced. 

But in my opinion specificity >>  exon exon junction. 

Dear Mohamed

exon junction or intron spanning primers are definitely the best as they target specifically RNA and not parent genomic DNA. Even if the genomic DNA is much larger is size and therefore will not realistically amplify under the time limits of PCR designed to amplify size limited qPCR products (100-300bp) nevertheless is can drain your principal qPCR reaction and under mine priming efficiency

Thus, exon spanning primers are best. In that regard primer BLAST and 2 sites by IDT (primer quest) and Roche (universal qPCR primer design tool) are excellent in my experience:

1. http://www.ncbi.nlm.nih.gov/tools/primer-blast/

2. http://www.idtdna.com/primerquest/home/index

3. https://lifescience.roche.com/webapp/wcs/stores/servlet/CategoryDisplay?tab=Assay+Design+Center&identifier=Universal+Probe+Library&langId=-1

In all 3 cases you will need to find your NCBI ref sequence (from NCBI Gen Bank) and for the IDT and Roche tools input the ref sequence as a FASTA file or simply type the Ref Seq accession # and you will get back exon spanning (qPCR) primers embedded in your ref sequence

Primer BLAST incidentally is particularly effective for making spliceform specific primers whereas Primer quest and universal primer design tolls are more direct

Sometimes, in my experience, exon spanning primers do not amplify as efficiently as internal exon primers. Thus you can perform qPCR with internal exon primers but in this instance make sure you have rigorously removed genomic DNA ny DNAse I digestion

Qiagens RNAse free DNAse I and PE (Ambion) Turbo rDNAse are particularly effective in that regard

1. https://www.qiagen.com/gb/shop/lab-basics/enzymes/RNase-Free-DNase-Set?cmpid=QVen2GADNase&kwid=%2bqiagen+%2brnase+%2bfree+%2bdnase#orderinginformation

2. https://www.thermofisher.com/order/catalog/product/AM2238

The latter in particular allows you to aggressively digest RNA for 1 hour at 37C without concomitant digestion of parent RNA, a problem with removing GDNA from total RNA when digesting @ 37C for > 30 min (with many DNAse I preps)

If you do decide to go down the exon spanning qPCR primer route - rather than internal exon primers + DNASe I digestion route (although the latter will work particularly for genes with large introns) find attached 2 x SOPs detailing how to use IDT primer quest and also primer BLAST 

Both exon junction and primer specificity has their own significance. Primer designed to exon junction ensures that it will target the cDNA not the DNA. if the intron between two exons is beyond amplification range. Thus it will not contribute expression profile due to amplification from genomic DNA. Where as non target amplification from pool of cDNA can eliminated by optimizing the primer concentration. So you can design the primer to exon junctions and achieve specificity by optimizing primer concentration.

Good luck.....

many thanks for all 

special thanks to Laurence for your shared files and detailed answer ... they really help