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Tips and tricks to design and test qPCR primers - advanced procedure

Design and test your own qPCR primers

 
1. Find the NCBI Gene page for the gene you would like to study. For example, for Human GAPDH.
 
2. Scroll down to the mRNA and Protein(s) section of the NCBI gene page and determine how many mRNA variants are listed:
3. Decide which mRNA variant(s) you would like to study.  If you would like to study only one variant, go to step 4a, otherwise skip to step 4b. 
 
4a. Use Primer-Blast to design primers specific for a single mRNA variant:
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).

Example of output aiming at designing PCR primers

Primer-Blast output when designing PCR primers

 

 

 

 

 

 

 

 

 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
4b. Use Primer-Blast to design primers to amplify regions from multiple mRNA variants of the same gene:

Example of output for sequence alignment (beginning and end of the sequences are only shown here)

Multiple Sequence Alignment - PCR primer design part 1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Multiple Sequence Alignment part 2

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Here, you can see that the two GAPDH mRNA variants have precisely the same sequences from position 203 to position 1401 of NM_002046.
These values are based on the clustal alignment above-described

Example of output aiming at designing PCR primers

Proposed PCR primers

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Here you can see that the proposed primers should amplify regions from multiple mRNA variants, but all will give a product of the same length and sequence.

5. Order the proposed Forward and Reverse primers (at least 2 sets) from a provider of pre-designed and pre-tested Primer Sets or of custom PCR primer synthesis.

Sample validation data for commercially-validated primers (amplification and melting curves, efficiency plots and EtBr-stained agarose gels demonstrating specificity). Here are examples of qPCR Primers for Human GAPDH (HQP006940) and murine GAPDH (MQP027158).

Predesigned and pretested qPCR primers for human and mutine GAPDH

 

 

 

 

 

 

 

 

 

 

 

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.