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Design SYBR Green qPCR Primers

Experimental protocol | Oct 31, 2013 Recommendations: +2

Authors

Mark Livingstone

Procedure

1) Find the NCBI Gene page for the gene you would like to study. For example, for           human GAPDH, the page is:  http://www.ncbi.nlm.nih.gov/gene/2597

2) Scroll down to the mRNA and Protein(s) section of the NCBI gene page and        determine how many mRNA variants are listed.  For example for human GAPDH,          there are two mRNA variants: NM_001256799 and NM_002046.

3) Decide which mRNA variant(s) you would like to study.  If you would like to only         study one variant, go to step 4a, otherwise skip to step 4b. 

4a) Use Primer-Blast (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) to design         primers specific for a single mRNA variant.

     A. In the box "Enter accession, gi, or FASTA sequence" paste the mRNA variant accession (for example: NM_002046).

     B. For PCR Product Size, set the Min=90 and the Max=300.

     C. Check the Intron Inclusion box: Primer pair must be separated by at least one intron on the corresponding genomic DNA (Note: Primer-Blast may fail to find primers that span an intron, so you may have to uncheck this box and search again, however, the resulting primers will also amplify genomic DNA, so DNAse digestion of mRNA should be performed prior to the reverse transcription step of qPCR using a good RNAse-free DNAse)

     D. Under Primer Pair Specificity Checking Parameter change the organism to whatever organism you are studing (e.g. Homo sapiens, Mus musculus)

     E. Check the box "Show results in a new window"

     F. Click "Get Primers". (See Image 1 for sample output).

     G. Confirm that the primers do not have any "Products on potentially unintended templates" in the output. (Now skip to step 5)

4b) Use Primer-Blast (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) to design         primers to amplify regions from multiple mRNA variants of the same gene:

     A. Use a clustal alignment site: http://www.ebi.ac.uk/Tools/msa/clustalw2/

     B. Change the tab from "Protein" to "DNA"

     C. Copy-Paste multiple FASTA sequences for the mRNAs you would like to study into the box.

             1. For example, open the two mRNA variant links NM_001256799 and NM_002046.

             2. At the top of the pages, click on the FASTA links.

             3. Copy the FASTA sequences and paste them into the Clustal box one after the other.

             4. Repeat for more mRNA variant sequences if desired, pasting the new sequence on the line following the previous one.

             5. Click "Execute Multiple Sequence Alignment"

     D. In the box "Enter accession, gi, or FASTA sequence" paste the mRNA variant accession (for example: NM_002046).

     E. Just to the right of that box, enter a range for the forward primer, From: 203, To: 1101. (Note: these values are based on the clustal alignment, see Image 2 and 3)

     F. For PCR Product Size, set the Min=90 and the Max=300.

     G. Check the Intron Inclusion box: Primer pair must be separated by at least                      one intron on the corresponding genomic DNA

     H. Under Primer Pair Specificity Checking Parameter change the organism to whatever organism you are studing (e.g. Homo sapiens, Mus musculus)

     I. Check the box "Show results in a new window"

     J. Click "Get Primers" (See image 4 for sample output)

5) Order the proposed Forward and Reverse primers (at least 2 sets) from a low-cost     provider of custom synthesis.

6) Primers will generally arrive lyophilized (dry) with instructions for how much water or TE buffer to add for 100uM solution.  Dilute the forward and reverse primers separately to 100uM with 1X TE Buffer, pH8.0.

7) Mix together 800uL Nuclease-Free Water 100uL forward primer and 100ul reverse primer for a final working stock of 10uM forward and reverse mixture. You will use this mixture at a final concentration of 0.4uM for qPCR.


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Comments: Showing comments 1-4 of 4
img Mark Livingstone
Thu, 1 May 2014 - 19:33 CET

Oh yes it is difficult to work with a "provisional" NCBI entry like this XM…I guess if you only have the cds, that's all you can work with. I didn't realize the C. albicans genome was not fully characterized. Somebody must have sequenced it by now. In general you can include all of the 5'UTR and 3'UTR unless you think there will be alternative translation initiation variants. Doing RT with just oligodT has its limits as you suggest. Some people use a combination of random hexamers and oligodT to avoid this problem. Others just use random hexamers. To see how efficient your DNAse treatment is, you really just have to run the RT reaction in parallel with an RT reaction where you have left out the RT enzyme. Then compare the two samples so you know just what signal comes from your RT.

img Amber Khan
Wed, 30 Apr 2014 - 17:14 CET

Hi Mark,
Thanks a lot for providing so much information. Mark, I am working on yeast Candida albicans (type strain SC5314). I am looking for six genes- EFG1, TUP1, GSH1, GSH2, SOD1 and ZWF1. Among them, two genes ZWF1 and GSH1 have (AFA I searched) not been assessed using qPCR. When I pick the primers e.g. for ZWF1, I get two sequences XM_718158.1 and XM_718347.1 gene/orf name of these I confirm from candida genome database, both are available as complete cds in ncbi. Moreover, I do not find any intron. I will have to design primers against cds only, but then which portion of cds (5'/3') should be included. The RT I am using can only produce cDNA upto13kb. That means I should focus 5'cDNA/3'mRNA, but where (?) I dont have any UTR available from either side? My mRNA cds is 1587bases long. As you suggested, then I'll have to run all +,- controls. I also should digest gDNA with RNase free DNaseI before stepping to reverse transcription. But I am not sure how efficient it will be?
Please could you assist.
Thanks and regards
Amber

img Mark Livingstone
Tue, 29 Apr 2014 - 19:29 CET

Hi Amber,
I'm fairly certain that primer blast combines fairly standard pcr primer design algorithms taking into account melting temperature etc. and also does a blast against other mRNAs in the species to test for specificity. I have a feeling it does not consider genomic DNA sequences. The thymidine is for the primer sequences and the cDNA that will be amplified. Certainly the RNA that will be reverse transcribed will have uracil instead. Generally these mRNA sequences NM_…. contain both the 5' and 3' UTRs. Sometimes the best forward or reverse primers will be in these, but it is usually good to have a fairly large intron between the forward and reverse primer to be certain that you won't amplify genomic DNA. What is your gene (species and gene name)? If you only know the cds and not where the exon junction complexes are you can still make good primers, but you won't be certain that you have an exon junction complex. In this case it will be good to run controls for genomic DNA, like +/- RT samples.

img Amber Khan
Sun, 27 Apr 2014 - 20:55 CET

Hi Mark,
Could you please explain how does this algorithm operate. Does the PRIMER BLAST use mRNA sequence (only 'cds') as the template for primer design (in that case it will be only used for making transcript specific cDNA in reverse transcription) or does it use gene exons (DNA) as template for primer design (but then in your protocol it is merely for selection purpose?)?
Why does NCBI refseq mRNA cds contains Thymine instead of uracil? Is it then DNA (sense/antisense?)?
PRIMER BLAST then blast primers with the refseq mRNA pool, what about the UTRs? My gene is only available as mRNA complete cds, and I want to make primers for quantitative (SYBR GREEN) PCR. Please could you assist.
Thanks and regards
Amber

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