Perfecta sybr green supermix with low rox

Quantitative PCR was performed on a Stratagene MX3000P qPCR machine by using PerfeCta SYBR Green SuperMix with Low ROX (Quanta BioSciences, Inc., Gaithersburg, MD). Twenty-five μl reactions were carried out containing half the reaction volume (12.5 μl) of SYBR, 12.5 ng cDNA, and forward and reverse primers at a concentration of 300 nM. Samples were run in duplicate at 95°C for 5 min, 40 cycles of 95°C for 30 sec/60°C for 1 min/70°C for 30 sec, followed by dissociation curves of PCR products to confirm primer specificity.
Samples were run across two plates and a common calibrator run on each confirmed low inter-plate variability (% CV = 8.25). No template controls were also run in duplicate on each plate and demonstrated no amplification. To quantify CB₁ mRNA expression, mean C_T values were determined between reaction duplicates for each sample. Mean CB₁ C_T values were then normalized to those of an internal reference gene (GAPDH) using the 2^{− CT}method (Schmittgen and Livak, 2008 (link)).

For all 79 males in studies 1 and 2, we extracted RNA from one testis using Trizol (Invitrogen, Carlsbad, CA, USA), treated 1μg of RNA with DNAse (Promega, Madison, WI, USA), and reverse transcribed it to cDNA with Superscript III (Invitrogen). All qPCR reactions were run on Strategene MX3000P using MxPro software (v.4.10, Agilent, Santa Clara, CA, USA) using PerfeCta SYBR Green SuperMix with low ROX (Quanta Biosciences, Gaithersburg, MD, USA). Samples were run in duplicate for each gene of interest (GOI) and a reference gene. We used the comparative Ct method (2^−ΔΔCt) that represents transcript abundance for each GOI as the fold difference in expression compared to a pooled standard (i.e. calibrator) and normalized by the reference gene (Livak and Schmittgen, 2001 (link)). The calibrator sample was derived from juncos collected during earlier pilot work. We used ribosomal protein L13A (RPL13A) as the reference gene because it has been reported to be one of the 10 best housekeeping genes for qPCR (de Jonge et al., 2007 (link)), and we confirmed that the populations did not differ in expression of this gene in the gonad. Further details on primers and our qPCR protocol can be found in ESM.

Control and mutant embryos isolated at 31–32 somite stage were used for these experiments. The regions containing either the left or the right pharyngeal arches 3–6 were dissected and stored in liquid N₂ until use. RNA was isolated using RNeasy plus micro kit (Qiagen) and cDNA was synthesized using SuperScript VILO cDNA synthesis kit (Life Technologies). The PCR was performed using PerfeCTa SYBR green supermix with low ROX (cat #95056-100, Quanta Biosciences) and Stratagene Mx3000P instrument (Agilent Technologies). The primers for detecting 18S RNA were 5′-GTAACCCGTTGAACCCCATT-3′ and 5′-CCATCCAATCGGTAGTAGCG-3′, the primers for detecting integrin α5 were 5′-CCTTTTTGGCTTCTCCGTGG-3′ and 5′-ACCACCTTGCAGTACACCTG-3′, and the primers for detecting Tbx1 were 5′-TCAAGGCTCCGGTGAAGAAG-3′ and 5′-TGGAACGTGGGGAACATTC-3′.