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RT-PCR primer design

designing primers primers Ensembl Ncbi RT-PCR

Best Answer doxorubicin, 05 August 2013 - 04:46 AM

It works well to just use NCBI gene:  http://www.ncbi.nlm.nih.gov/gene database in combination with Primer-Blast:  http://www.ncbi.nlm.nih.gov/tools/primer-blast/

 

Search for your gene name, scroll down to see how many mRNA variants (NM_......) are listed. Pick the one that you want to detect by qPCR, then just copy-paste the accession number (NM_....) into the search window of Primer-Blast.

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#1 endlessdreamer

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Posted 05 August 2013 - 03:31 AM

Hi all,

 

I'm designing some primers for RT-PCR (qPCR) and to do this, I find the mRNA sequence of the gene of interest on Ncbi and Ensembl. Then I copy the sequence from ncbi into Primer3 to select primers and find which exons the primers sit by using Ensembl. The trouble is that I can't always find matching mRNA sequences in Ncbi and Ensembl so I can't always find which exons the primers sit in (which is important to determine if there is genomic contamination). Please can someone offer some guidance?

 

Many thanks!



#2 doxorubicin

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Posted 05 August 2013 - 04:46 AM   Best Answer

It works well to just use NCBI gene:  http://www.ncbi.nlm.nih.gov/gene database in combination with Primer-Blast:  http://www.ncbi.nlm.nih.gov/tools/primer-blast/

 

Search for your gene name, scroll down to see how many mRNA variants (NM_......) are listed. Pick the one that you want to detect by qPCR, then just copy-paste the accession number (NM_....) into the search window of Primer-Blast.



#3 endlessdreamer

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Posted 05 August 2013 - 04:53 AM

Thank you! I just figured out another way too :)



#4 pcrman

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Posted 05 August 2013 - 01:11 PM

My two cents: First obtain the refseq number in NCBI, then use Primer-Blast to design primers.



#5 memari

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Posted 05 August 2013 - 07:20 PM

Top Ten Pitfalls in Quantitative Real-time PCR Primer Probe Design and Use

https://babakmemari....design-and-use/


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Babak Memari

#6 memari

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Posted 05 August 2013 - 07:26 PM

or go to http://www.origene.com

 

and click on "Register To View Sequence" and write an email even a fake one to see sequence:

like:

http://www.origene.c...r/HP220445.aspx


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Babak Memari

#7 endlessdreamer

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Posted 06 August 2013 - 03:50 PM

Thank you very much for all the links and guidance. Fab ideas. I'm only now worried that the primers I've designed for qPCR will form products that are greater than 150bp in length (up to 240bp but most are ~170bp). Anyway, will see how it goes.



#8 doxorubicin

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Posted 14 November 2013 - 12:39 AM

I've recently written a very complete protocol for designing primers for SYBR green qPCR, and posted it on our alternative careers group page:

 

https://sites.google...wn-qpcr-primers

 

It even explains how to do clustal alignments and use this information in Primer-Blast in case you want your primers to detect multiple mRNA variants of the same gene that will all give products of the same size.  I personally always set my amplicon sizes to be between 90 and 300bp, and this has worked well for me hundreds of times.  







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