I am trying to perform a monochrome multiplex qPCR (MMQPCR) for assessing telomere length (but advice about any MMQPCR technique would be fine). I am following Cawthon et al. Nucleic Acids Res. 37:e21. Briefly, they use a Master Mix with SYBR, and two pair of primers (telomeric and a single-copy gene). The method relies in designing such primers that the products have different melting temperatures (by using GC clamps), and reading the fluorescence at two temperatures in each cycle. The first signal acquisition detects only telomere product at early cycles, with the reference gene (a single-copy gene) is still at baseline, and the second acquisition, at higher temperature, reads only the product of the reference gene (because the telomere product is melted).
I am using a StepOne Plus real-time PCR system (Applied Biosystems), and I have no clue how to get these two reads to work. I tried once and got only one read, possibly the second one. However, the melting curve confirms that I have two products (very similar to the melting curve reported by Cawthon et al.!). In these study, they use the "Bio-Rad MyiQ Single Color Real-Time PCR Detection System", with the MyiQ software (Bio-Rad iQ5 2.0 Standard Edition Optical System Software).
Maybe the StepOne software does not allow to register two readings per cycle?
Other method is described by Jiao et al. in Analytical and Bioanalytical Chemistry 403:157-166 (2012). Here, they use a different strategy. They perform 20 cycles with a melting stage at 85 °C, thus melting the telomere product, but not the reference gene product. Then, they perform other 20 cycles, but this time melting occurs at 94 °C and extension-reading at 85 °C, when telomere products are melted, but reference gene products are not. I want to try this approach, but again, I have no idea if the StepOne Plus will discriminate readings in different cycling stages, or if the second stage would overwrite the first one. Jiao et al. also used a Bio-Rad device (Bio-Rad iQ5 96-Well Real-Time PCR Detection System).
I am contacting the AB guys, but I would greatly appreciate any advice from researchers using MMQPCR in StepOne devices. Thank you!
OK, received response from AB (quick and very nice). No way, the StepOne Plus cannot read twice in the same cycle (it just sums up both fluorescence readings).
I will try the method by Jiao et al. and then process the raw data "by hand", just to see if it works.
Yes, it works. Since it is not possible to carry out by multiplex, I am performing the analysis in twin plates (telomere primers vs. single-copy gene primers).
No problem, I can share it here. We were also getting amplification in the blank wells (water instead of DNA). We are now using the Brilliant III real-time PCR kit from Agilent, and it is working OK (I think that the issue was that the previous kit was not adequate for this experiment). We perform an annealing-extension step at 60 °C for 30 s and a denaturing step at 95 °C for 15 s, about 35 cycles, and a melting analysis afterwards.
- Badges
- Science method