So the lab that I work in generates a lot of AAVs and we titer them via QPCR. However, I've been noticing that the entire time, the efficiency has always been very low (anywhere from 40-80%) with certain primers. It's apparently been that way since before I started and before the person who trained me started so I'm trying to troubleshoot this.
We typically use WPRE as the element we titer with. My PI has always said that those primers are the most reliable in his experience and we've generally kept to that except in cases where the viral genome does not contain WPRE.
I've tried everything I can think of from linearizing the plasmid DNA for standards, running gradients to be sure that the annealing temperature is correct, changing the annealing/extension time from 30 seconds to 1 minute, and played with primer concentration a tiny bit. I'm beginning to believe that there is some sort of secondary structure in the amplicon that is causing this poor efficiency or the machine (Bio-Rad CFX Connect) needs to be calibrated. I use Bio-Rad's iQ SYBR Green Supermix.
My protocol generally has a 3 minute 95C initial activation, 10 second 95C denaturation, and a 30 second 55C annealing/extenstion time with 40 cycles. I've done melt curves and am getting specific products which is good, but the efficiency is low no matter what I'm changing or doing. My r^2 values are also generally good (between 0.98-0.99) and my replicates for my samples are also typically close in Cq value. I just can't seem to get the standards right for some reason. I use a 1:3 dilution and have considered altering that to a 1:5 to see if that improves things but I don't really see why that would make a difference.
Does anybody have any ideas, tips, or tricks that can help me troubleshoot this protocol?
You're right. WPRE is rich in GC and forms a strong secondary structure. It's the last place I'd ever put a primer. Not surprised it's inefficient. Try to find a GC/AT balanced region, and follow good practices for primer design. NCBI's primer tool among many others does this automatically.
Thank you John Schloendorn! Do you know of a way to analyze secondary structure? I tried using mfold but it doesn't seem to exist anymore and I tried using the one on IDTs website but I don't really understand what I'm looking at.
Do you have any tips for potentially getting around this secondary structure issue apart from choosing another element in the sequence to titer with? I have less efficiency with other primers as well. I probably just need to run a ton of gradients to see if the annealing temp is in fact correct.
If the primer efficiency is low that suggests that the primer is not able to bind the template properly or as you speculate secondary structure inhibits primer binding. The way I would have to troubleshoot is doing qPCR at two different temperatures, 51 and 60. If the primer is not able to bind properly then lowering the Tm would help whereas if the secondary structure is causing a problem then increasing Tm might help.
In IDT, if you are checking for primer-dimer or hairpin structure of the primer then the del G value is of -10 or < -10 would suggest the primer is not going to bind properly with the template.
- Badges
- Science method