Empirical evaluation of humpback whale telomere length estimates; quality control and factors causing variability in the singleplex and multiplex qPCR methods

Table 1 Experimental setup of the four qPCR assays for relative telomere length estimation

Reaction conditions					Primers		Standards			Telomere		Reference
Assay	Platform	Type	Master mix	Duration	Tel	Ref	Batches	Dilutions	LDR	Cq	NTC	Cq	NTC
I	ABI	Singleplex	Commercial	21	CW2006	BA 1998	7	5	4.5-72.0	17.6	34.9	33.5	40.0
II	RG	Multiplex	RC 2009	6	RC 2009	RC 2009	4	4	2.2-60.0	15.7	19.4	39.9	27.9
III	RG	Singleplex	RC 2009	5	RC 2009	RC 2009	4	4	2.2-60.0	14.1	15.5	37.4	36.2
IV	RG	Multiplex	Commercial	6	RC 2009	RC 2009	4	5	4.5-72.0	13.9	27.2	34.3	31.1

Duration = number of days it took to generate the data once assays had been optimized; BA = Bérubé and Aguilar [38]; CW 2006 = Callicot and Womack [52]; RC 2009 = Cawthon [24]; Batches = number of separate qPCR runs (plates); Dilutions = number of dilution steps; LDR = linear dynamic range (ng/reaction); Cq = the average Cq of the serially diluted standard with the lowest amount of DNA; NTC = the lowest Cq value observed for a NTC.

ISSN: 2730-6844