Dear David,

You may take a look the following link, https://www.genscript.com/ssl-bin/app/primer

or

I. First get the cDNA sequence of my gene from NCBI. I only use Refseq if available. RefSeq represents the most reliable and unique cDNA sequence for a gene. Because searching the nucleotide database is not effective (you may get hundreds of sequences for a gene, most of them are irrelevant) so I will go to Entrez Gene database (previously LocusLink) at http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene to find the gene of my interest, then scroll down to the NCBI Reference Sequences (RefSeq) section to find the RefSeq mRNA sequence whose accession number usually looks like NM_xxxxxxx. Then, click the sequence link which will lead to the actual sequence from GenBank Nucleotide sequence database.

2. Copy the sequence to a program to design primers such as Primer3 at http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi. Just accept all default parameters provided by the program and design primers. Choose the pair with highest score.

3. Copy the sequence corresponding to the amplified region by the primer pair you have chosen and then go to UCSC genome browser at http://genome.ucsc.edu/cgi-bin/hgBlat and paste the sequence there and do a BLAT.

4. Check if the amplicon spans any introns by view the detailed alignment of the query sequence against genomic sequence.

5. Or do a BLAT using the whole cDNA sequence first to know where exon-exon junctions (on cDNA) are and then design primers by specifying a target region corresponding to the junctions

6. Another purpose of Blatting amplicon sequence against genome database is to check if the amplified fragment has any homology with other genomic regions. The Blat results will tell that.

7. There are some other places you can also check gene structure such as NCBI Aceview at http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/index.html and Ensembl at http://www.ensembl.org. (Collected)

Best of luck................

Http://www.protocol-online.org/biology-forums/posts/2706.html

please see the link.

Dear David,

You may take a look the following link, https://www.genscript.com/ssl-bin/app/primer

or

I. First get the cDNA sequence of my gene from NCBI. I only use Refseq if available. RefSeq represents the most reliable and unique cDNA sequence for a gene. Because searching the nucleotide database is not effective (you may get hundreds of sequences for a gene, most of them are irrelevant) so I will go to Entrez Gene database (previously LocusLink) at http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene to find the gene of my interest, then scroll down to the NCBI Reference Sequences (RefSeq) section to find the RefSeq mRNA sequence whose accession number usually looks like NM_xxxxxxx. Then, click the sequence link which will lead to the actual sequence from GenBank Nucleotide sequence database.

2. Copy the sequence to a program to design primers such as Primer3 at http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi. Just accept all default parameters provided by the program and design primers. Choose the pair with highest score.

3. Copy the sequence corresponding to the amplified region by the primer pair you have chosen and then go to UCSC genome browser at http://genome.ucsc.edu/cgi-bin/hgBlat and paste the sequence there and do a BLAT.

4. Check if the amplicon spans any introns by view the detailed alignment of the query sequence against genomic sequence.

5. Or do a BLAT using the whole cDNA sequence first to know where exon-exon junctions (on cDNA) are and then design primers by specifying a target region corresponding to the junctions

6. Another purpose of Blatting amplicon sequence against genome database is to check if the amplified fragment has any homology with other genomic regions. The Blat results will tell that.

7. There are some other places you can also check gene structure such as NCBI Aceview at http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/index.html and Ensembl at http://www.ensembl.org. (Collected)

Best of luck................

Hi David

Mohammad have explained it very well. ....you can follow any standard manual for Rt-PCR and use the information for your organism from the databases for effective protocol designing

bye

Genescript is providing free software

I don't know with which real-time PCR machine you want to run the PCRs, depending on this you have to design your primers. Machines like the ones from Applied Biosystems normally go for a 2-step-PCR (combining annealing and extension step) and therefore primer conditions are different than from 3-step-PCRs. If there is a specific software available for you real-time PCR machine I would prefer to use this software for primerdesign.

And independent with which software you design the primers - you have to crosscheck the specifity, what I do with the BLAST tool you find on the ncbi-webpage: http://www.ncbi.nlm.nih.gov/tools/primer-blast

Hi David,

you can also use the primer express software for quantitative real timeprimer designing.

I totally recommend this tool for qPCR primer designing, besides Pennisetum glaucum is included in their list of species. Give it a try!

http://www.quantprime.de/

Even if you are not interested in amplifying from RNA but only from DNA (for determining copy number for example), you can choose not to restrict primers in exon-intron junctions.

Good luck!

I designed all my primers using Roche's tool: http://www.roche-applied-science.com/webapp/wcs/stores/servlet/CategoryDisplay?catalogId=10001&tab=&identifier=Universal+Probe+Library&langId=-1#tab-3

Thank you Dr. Carl Fortin

I have followed your link primer balst and I have found very ideal primers pairs with no hairpin, dimers etc

I recommend everyone to follow that link for rt PCR primers

Step wise for designing RT Primers for specific gene:

1. Search for you gene through the following link

https://www.ncbi.nlm.nih.gov/gene

2. copy FASTA file and past at following link

https://www.ncbi.nlm.nih.gov/orffinder/

3. copy FASTA file of exact and correct ORF sequence of the gene and past at follwing link

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

4. Check for annealing Temperature by Tm calculator at following link

https://www.thermofisher.com/us/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/thermo-scientific-web-tools/tm-calculator.html

select you Master Mix and GC % should be greater than 50% for optimal Annealing.

5. And the Order from a company.

you can use this location

http://pcrsuite.cse.ucsc.edu/cDNA_Primers.html

you will find here good tips:

https://www.youtube.com/watch?v=XHdRW9xxgO4

Thank you all, i find genescript quite simple and useful.