low qPCR efficiency

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estaunton's picture
low qPCR efficiency

I've seen some people post about this problem, but I too am having issues with low qPCR efficiency. I'm trying to develop a method for quantification of the A subunit of a functional gene called hydrazine synthase (hzsA). The primers I am working with were published in January 2012 and are hzsA_1597F ( 5′-WTYGGKTATCARTATGTAG-3′ ) and hzsA_1857R ( 5′-AAABGGYGAATCATARTGGC-3′ ).
 I created my standard using the TOPO TA cloning kit for sequencing, digested the plasmid with EcoRI, and did a purification using QIAGEN Nucleotide removal kit. I quantified the DNA using a NanoDrop fluorospectrometer with the Quant-iT PicoGreen dsDNA Assay Kit.
 I ran 10X serial dilutions of the DNA and used QIAGEN Quantitect SYBRGreen qPCR mastermix with 0.5 uL of 25 mM MgCl2 stock solution and 0.5 uL each of 15 uM forward and reverse primers in a 25 uL reaction. This reaction gave me a 68% efficiency. The melting temperature was 55 degrees C.
 I did a preliminary experiment with 3 of my standards (10^6, 10^5 and 10^4 copies/uL) and increased the Mg concentration to 1 uL of 25 mM stock and got 95% efficiency, but when I ran the entire curve with the same conditions, I got about 78% efficiency.
 Does anybody have any idea why my efficiency is so variable? And what can I do to stabilize it?

Ivan Delgado
Ivan Delgado's picture
Hi estaunton,

Hi estaunton,

There are a number of reasons why your qPCR efficiency is that low: 

1. The PCR is not working. A 95% efficiency is actually not that good. It may sound harsh, but anything below 98% is questionable. 

2. The fact that you get higher effiiciency with a few standards than when you include all standards is likely due to your assay only working with the few standards that give you a higher efficiency. In other words, likely the standards that are making your efficiency lower contains either too much or too little DNA and as a result cause the PCR not to work (and decrease your efficiency). 

3. I think it is a good idea to use published primers, but the primers you listed look questionable. There is no reason why primers that have all sorts of "weird" bases would not work, but one of my first concerns about your assay not working would be that your primers have bases such as "W", "Y" and "K". If at all possible I would design at least 2 or 3 sets of primers and test those to see if you continue getting efficiency problems.

4. Yet another possibility is linked to the primers. The "weird" bases I mentioned in point 3 above basically means that your primers are synthesized containing different bases at those positions. In other words when you synthesize these primers you get a collection of primers containing many different bases (one primer having an A at position W and another primer in the same primer solution having a T at that same W position). This could easily be leading to the generation of more than one amplicon and thus give you unreliable efficiencies. 

I hope this helps,