Hi Dharitri Borah

You can use the same rules for qPCR primer design as you do for "normal PCR" just shoot for amplicons between 75-150bp (max. 250 bp) if possible.

here some informations:

http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html

Hi Dharitri Borah

You can use the same rules for qPCR primer design as you do for "normal PCR" just shoot for amplicons between 75-150bp (max. 250 bp) if possible.

here some informations:

http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html

Also,

make sure you validate your new primers after they arrive by performing a standard curve using a template that is similar to your experimental samples to determine the dynamic range.

Any of these are nice:

I hope that these articles and links will be useful.

Primer3 - http://primer3.sourceforge.net/

Also,,,,

First, you will need a primer designing software.

Free: NCBI primer search (using primer3 algorithm)

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

Pay: Premier biosoft

Some information of primer design:

http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html

Different between PCR/qPCR primer:

Product size: Recommended qPCR product size is preferably less than 200bp

PCR product size is limit by the DNA polymerase

Intron: qPCR primer for gene expression in eukaryotic cell. Primer spanning exon-exon junction prevent false amplification of contaminated genomic DNA

Probe: Software capable of designing probe is required in probe base qPCR

Default Primer design parameter is preset in the primer search software.

All that is required, insert your template sequence.

Check the primer length, Tm, product size, specificity check....

Important point:

Correct template: DNA or RNA input sequence

Primer binding location: If a particular region is needed to be amplified

Some understanding on the primer designing parameter so that a modification of the parameter is possible if there are no result available after primer search.

Primer dimer avoidance by looking at the primer-primer interaction activity.

A blast search of the selected primer give you a rough idea on its specificity.

MH

Thank you very much to all for your helpful suggestions. I have a doubt. I have come across a few published articles with q-pcr primers with target gene. But, when I blasted these primer sequences, it was not matching with the organism on which the study was conducted, except one of 10. The match was not even for the genus. Is it possible select some other organism when gene information is not available for the particular strain?

First I agree with all above comments about designing your primers.

Second, is the organism you are studying fully sequenced? If not that may be the reason you only identified one positive match via blast. It is possible, that that lab has their own sequencing data for that organism and they have yet to make the data publicly available but they therefore know the sequences for the genes of interest. If they don't have any sequencing data then they are likely using sequences of closely relative species, assuming the gene of interest is conserved, and if they have published successful qPCR data, you would assume the sequences are validated.

If the organism is sequenced, then during your blast search of the primer sequences, you can choose in the, choose search set, to pick an organism. Therefore it will only blast against known sequences for that organism. Thereby filtering out any other unrelated organism.

Hope that helps with your issues.

Not finding the published primers in BLAST can be due to a couple reasons.

The authors may have used "intron spanning" primers that anneal to the cDNA and not gDNA. These wouldn't show up as having a match to the genome, but only to the transcriptome.

The BLAST algorithm is optimized for longer sequences on the default settings. You will have to choose the "shorter, less conserved" option to get better results from a search of primer sequences.

If you don't reverse-complement the reverse primer, the algorithm will not be able to search efficiently for the reverse sequence.

So, specify the organism, use the transcriptome (if available), and choose settings that work well for short sequences. As Matthew de Cruz pointed out, this all assumes that the organism in question has been sequenced. You could always contact the lead author with any questions about the genome of your organism.