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Test a range of annealing temperatures. Depending on the qPCR results, the annealing temperature should be increased or decreased in 2-3oC increments. This can be done in a single experiment using a thermal gradient. Alternatively, a range of annealing temperatures should be tested using multiple qPCR experiments.
Thank you B.Dhungel, from Gallipoli Medical Research Foundation (GMRF), Greenslopes Private Hospital, University of Queensland for such a great review.
I am really impressed by the reproduciblity of the Bioline Isolate II RNA minikit, the cDNA sysnthesis and the Sybr-Lo Rox mix in qPCR. We consistently get high concentrations of pure RNA with 260/280 reading 2 when measured with the nanodrop.
We have seen that the cDNA synthesis kit from Bioline is able to synthesize high quality cDNA even from low starting RNA concentrations when analyzing the qPCR results.
Working with a new qPCR target
When looking at a new target of unknown expression level, the amount of RNA required to detect the target of interest depends on the abundance of the target in each sample. For high-copy-number transcripts you may be able to detect in as little as 10 pg, while for low-copy-number transcripts you may require more than 100 ng.
With all new targets (new sets of primers or new qPCR kits), a 10 fold serial dilution should always be run, to validate both the slope and the limit of detection (LOD) (the highest Ct value observed for a truly positive sample, as verified by melt-curve analysis). Only a single peak, which represents the specific PCR product, should be observed on the melt curve, the presence of other peaks indicates the presence of primer–dimers and/or nonspecific PCR products which can contribute to a stronger fluorescence signal and an earlier Ct, but will not be as sensitive.