qPCR primer design - RNA template

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Mmarturo
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qPCR primer design - RNA template

Dear all,

I am designing qPCR primers for some genes in drosophila melanogaster.
I am currently using NCBI tool which uses Primer3 and BLAST to find the primer pairs from the refSeq records. I am using the thermofynamic secundary structure aligments to "avoid" harpins, dimers and primer-dimers in Primer3 tool, however I am later checking them manually with OligoAnalyzer 3.1 tool (http://eu.idtdna.com/sessionTimeout.aspx) to make sure harpins have at most DG=-3, self-dimer is less than DG=-5, cross-dimer (sense and antisense primers homology) to be less than DG=-5, and so on.

My question are:
1 - If the template we will use for qPCR is RNA, do I have to search primers that span an exon-exon junction? 

2 - Since we will amplify from RNA template, do I need to do the reverse complement of the designed primers found from refSeq (mRNA)? See attached file with an example, for a gene in positive strand, the forward primer matches to the gDNA, and the revesere primer is the reverse complement of the gDNA. And the opposite for a gene in the reverse strand. How should I order my primers?

Sushma Dagar
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 Your procedure of designing

 Your procedure of designing primer is perfect.
yes you should design primers at exon-exon junction.

Sushma Dagar
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You should order your primer

You should order your primer in the same sequence which you are designing. You don't need to do reverse complement because when we design primers, using ncbi tool, we don't get primers complimentary to mRNA (primers will be having the same sequence as that of RNA). And While doing qPCR, we convert our RNA into cDNA, So ultimately primers will be complimentary to the cDNA ( as your image shows that primers are binding to gDNA). 

Mmarturo
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 Thank you very much for your

 Thank you very much for your comments.

I'm designing the primers with the constraint that forward primer must span within the first exon, so it is in some cases very difficult to find a pair of primers with a product size of 50-200 bps and spaning exon-exon junction for the reverse primer. Is exon-exon junction a must or just increases the efficiency?

You mentioned that while doing qPCR we convert our RNA to cDNA, is this always the case even if we want to amplify from RNA?

Thank you again for your comments,

Sushma Dagar
Sushma Dagar's picture
 Designing primers on exon-

 Designing primers on exon- exon junction increases the efficiency and avoid the chances of non specific amplification of genomic DNA. But it is not necessary. Since your criteria is very much specific so its very difficult to get primer on exon exon junction. 

As far as I know, yes you need to convert your RNA to cDNA for doing qPCR. even if you are converting RNA to cDNA, whatever results you will get will be based on your starting RNA.
But I am not 100% sure.
1. Stability of RNA is always a matter of concern even if you are storing at -80 C. So its always  good to convert your RNA into cDNA and store.
2. While doing qPCR, we use syber green which contains DNA polymerase, dNTPs etc. but not reverse trancriptase. 

Mmarturo
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 Thank you again for your

 Thank you again for your clear and useful information "Science rocks"!

Sushma Dagar
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 All  the Best..

 All  the Best..

Hazman77
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 I would like to ask about

 I would like to ask about the difference between designing primers on the positive strans and negative strand cDNA for qPCR?
thanks and all the best

Ivan Delgado
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If I understand your question

If I understand your question correctly, there is little difference between primers designed on one strand versus the other, mainly since the two strands are just reverse complements of each other.

Now, if by designing primers you mean doing it by hand instead of using an algorithm, then there is a good chance you will have different primers since you will not be using the criteria for primer design included in the algorithm.

Hope this helps.

Hazman77
Hazman77's picture
 hallo,  how you can analysis

 hallo,  how you can analysis your data statistically. If you have only two replica? 

Ivan Delgado
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As far as I know you can only

As far as I know you can only perform statistical analysis if you have 3 or more biological replicates.

Replicates, i.e. technical replicates (same sample run twice or more), is something else. That can give you an idea of how much variation there is in the way your experiment is performed, but it does not give you any information about the significance of the biological question you are studying.

Hazman77
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 thanks alot for your reply

 thanks alot for your reply