4.3. Monochrome Multiplex Quantitative PCR (MMQPCR) to Measure Relative Telomere Lengths

MMQPCR was conducted following the protocol outlined in Vaquero–Sedas and Vega–Palas [52] with minimal modifications. Primer sequences for genes used in this study are shown in Table 2, including primers for the single copy gene, PP2A, and for the telomere sequence [52,53]. Oligos were synthesized by MilliporeSigma (Burlington, MA, USA) and resuspended to a concentration of 100 μmol/L upon arrival. Standard curves were created for each primer pair by pooling six aliquots of DNA isolated from a single clone of the almond cultivar Nonpareil, and performing successive dilutions to 20 ng/μL, 10 ng/μL, 1 ng/μL, 0.5 ng/μL, and 0.25 ng/μL. Reactions were carried out in triplicate for each primer by concentration combination.

Oligos used for all Monochrome Multiplex Quantitative PCR (MMQPCR) and quantitative reverse transcriptase PCR (qRT-PCR) studies.

Isolated DNA from the age cohort samples was diluted to 20 ng/μL. Multiplex reactions were carried out in sextuplicate for each replicate within the age cohorts in a 10 μL volume using QuantaBio PerfeCTa SYBR^® Green SuperMix (Quanta Biosciences, Beverly, MA, USA) (2×), forward and reverse primers (100 nmol/L each), and 20 ng template DNA according to the manufacturer’s instructions. Reactions were performed in a Bio Rad C1000 Touch Thermal Cycler (Bio Rad Laboratories, Hercules, CA, USA) using the following program: initial denaturation at 95 °C for 3 min followed by 2 cycles of incubation at 94 °C for 15 s and annealing at 49 °C for 15 s; telomere and PP2A amplicons were generated following 35 cycles at 95 °C for 30 s, 59 °C for 1 min, 72 °C for 30 s, 84 °C for 15 s, and 85 °C for 15 s; final incubation at 72 °C for 1 min. Melting curve analysis was performed at a temperature range of 74–85 °C for both primer pairs to ensure no non-specific amplification.