Primer and probe sequences and conditions used for real-time RT-PCR for analysis of mammary gene expression and milk production in Norwegian dairy goats fed concentrates supplemented with palm oil fatty acids or rapeseed oil throughout lactation
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Total RNA was prepared through the homogenization of approximately 20 mg of biopsy tissue in 0.35 ml of TRIzol Reagent (Invitrogen Life Technologies, Cergy Pontoise, France), followed by isolation using the Pure Link RNA mini kit isolation system (Invitrogen, Carlsbad, CA, USA), according to the instructions of the manufacturer. Potential contaminating genomic DNA was removed through a DNase treatment step (RNase-Free DNase Set #79254, Qiagen, Hilden, Germany). RNA content were determined by measuring absorbance at 260, 280 and 320 nm using a NanoDrop™ (ND-1000 spectrophotometer; NanoDrop, Labtech, Palaiseau, France). RNA integrity was determined using a 2100 Bioanalyzer (Agilent Technologies, Massy, France) and was 7.6 (SD 0.79) on average. Reverse transcription was performed from 2 µg of purified total RNA isolated from the mammary samples using the High-Capacity RNA-to-cDNA kit containing dNTPs, random octamers, and oligo dT-16 (Applied Biosystems, Foster City, CA) in a final volume of 20 µl. The samples were stored at -20°C. The mRNA abundance of the following 19 candidate genes was measured via quantitative RT-PCR: FASN, G6PD, LPL, CD36, FABP3, GLUT4, SCD1, SCD5, GPAM, LALBA, CSN1S1, CSN2, CSN3, XDH, MFGE8, CASP8, ELF3, SREBF1, PPARG1. To account for variation in RNA integrity, RNA quantification and cDNA synthesis, the mRNA abundance was normalized using the geometric mean of three housekeeping genes MRPL39, UXT and EIF3K, which were identified as suitable internal controls for caprine mammary tissues among several tested (Bonnet et al., 2013) with their stability verified using GeNorm software. The mRNA abundance was quantified in duplicate via real-time quantitative RT-PCR using the StepOnePlus TM real-time PCR system (Applied Biosystems, Courtaboeuf, France), SYBR Green dye (TF Power SYBRGreen PCRMasterMix) or a fluorescent TaqMan probe (TF TaqManFast Universal PCR Master Mix), according to the manufacturer’s instructions (Applied Biosystems) and with specific primers and probes (Supplemental Table S1). Specific primers and probes were designed on a consensus cDNA fragment among species. Briefly, for SYBR Green technology, after an initial denaturing step (95°C for 15 min), the PCR mixture was subjected to the following 2-step cycle repeated 40 times consisting of denaturing for 15 s at 94°C and annealing and extension for 45 s at 58 or 60°C (depending of the primer pairs). Real-time PCR based on TaqMan probe technology was performed under the same conditions but the annealing for primer pairs was always 45 s at 60°C. PCR efficiency was 88.4% (SD 6.24) for the 19 target genes and 91.4% (SD 0.83) for the three reference genes. The abundance of candidate gene transcripts was expressed as the mRNA copy number relative to the geometric mean of the three housekeeping genes to account for variations in RNA integrity, RNA quantification and complementary DNA synthesis.