Reducing background in qPCR with SYBR green

2 posts / 0 new
Last post
o2bake
o2bake's picture
Reducing background in qPCR with SYBR green

 I'm working on a qPCR protocol where I'm trying to get optimal parameters for 9 different primer pairs per 96-well plate using bacterial genomic DNA as template. I'm currently using SYBR green for detection, since hydrolysis probes would be a little pricey at this point. My problem is that I'm getting too much background in some of my primer pairs' no-template controls, typically at about the same Ct as the 10 fg level of their respective standard curves. This is equivalent to around 2 cells worth of gDNA, which isn't a lot, but tell that to my boss. Anyway, I'm looking for some ideas for knocking down the background, keeping in mind that I have to strike a balance for all 9 primer pairs.

Ivan Delgado
Ivan Delgado's picture
 

 
Hi o2bake,

Here are my suggestions regarding your situation: 

1. A signal at 10 fg is considered statistically insignificant as long as it is at least 1,000-fold less abundant as the lowest signal you are considering in your data. For example, you can use any data point above 10 ng (nanograms) in your standard curve if the Ct of your 10 ng point is the same as the Ct of your NTC. The rationale here is that a 1,000-fold difference is only 0.1% of your signal, which for all intended purposes is not significant (your NTC's signal at 10 fg would only account for 0.1% of your 10 ng point's signal). 

2. If somehow you cannot convince the person in charge that getting a signal at 10 fg is perfectly fine (as long as it meets the requirements in #1 above), then you should design other primer pairs and identify one that does not give you that background signal. In my experience, when designing SYBR assays, it is a good idea to test at least 3 primer pairs to identify one that works the best. 

3. The background signal you are detecting is only applicable if the amplification curve is real. When looking at the amplification plots, determine if the low signal at 10 fg is actually real (a clear amplification curve), or if it is just the tail end of amplification (which could be real or it could not be). One way to assess if this 10 fg signal is real is by raising the threshold line by 0.1 units or more. If your standard curve's values are pretty much unchanged, and the NTC's 10 fg signal disappears, then you can assume that the NTC's signal is insignificant. 

Cheers