Thermal cycler dice real time system 3 tp950

Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions. Purity and concentration of the RNA were analyzed by NanoDrop (DeNovix, DE, USA). Total RNA (2 µg) was reverse transcribed into cDNA using the QuantiNova reverse transcription kit (Qiagen, Germany). Real-time quantitative polymerase chain reaction (qRT-PCR) was conducted using the Thermal Cycler Dice Real-Time System III (TP950) (TAKARA, Japan) with the GoTaq^® qPCR Master Mix (Promega, WI, USA) under the following conditions: 40 cycles of denaturation 15 s at 95 °C and amplification 1 min at 60 °C. Data were analyzed by the 2^−ΔΔCT method, and gene expression was normalized to β-actin. Primer sets were: mouse NFATc1 (5′-CCGTTGCTTCCAGAAAATAACA-3′ and 5′-TGTGGGATGTGAACTCGGAA-3′), mouse TRAP (5′-ACTTCCCCAGCCCTTACTAC-3′ and 5′-ACATAGCCCACACCGTTCTC-3′), mouse cathepsin K (5′-CTTCCAATACGTGCAGCAGA-3′ and 5′-GTGCTTGCTTCCCTTCTGG-3′), and mouse β-actin (5′-AGCCATGTACGTAGCCATCC-3′ and 5′-CTCTCAGCTGTGGTGGTGA-3′.

Total RNA from testes was extracted using ISOGEN reagent (Nippon Gene, Tokyo, Japan), treated with RNase-free DNase I (Qiagen, Hilden, Germany), and reverse transcribed using an oligo(dT)18 primer with ReverTra Ace (Toyobo, Tokyo, Japan). Quantification of complementary DNA (cDNA) from each testis was measured using Bio Photometer (Eppendorf, Hamburg, Germany), followed by dilution with nuclease-free water. Aliquots of diluted cDNA were stored at − 20 °C.
Levels of target gene expression were determined using 100 ng of template cDNA per reaction, and quantified on either a Prism 7000 Real Time PCR System (Life Technologies, Carlsbad, CA, USA) or a Thermal Cycler Dice Real-Time System III TP950 (Takara Bio, Shiga, Japan) with appropriate probes or primers. The expression of each gene was normalized to glyceraldehyde 3-phosphate dehydrogenase (Gapdh) levels and quantified by using the 2^−ΔΔCq method [29 ].

The expression of genes encoding the antioxidant enzymes superoxide dismutase 1 (Sod1), catalase (Cat), and glutathione (GSH) peroxidase 1 (Gpx1), as well as enzymes involved in GSH synthesis, namely GSH reductase (Gsr) and GSH synthase (Gss), was detected by qPCR. Additionally, we examined gene expression of Cyp11a1 encoding the rate-limiting enzyme P450scc for steroid biosynthesis. Cycling conditions were as follows: 95 °C for 2 min, and 40 cycles of 5 s at 95 °C and 30 s at 60 °C. Reactions were performed in triplicate on a Thermal Cycler Dice Real-Time System III TP950 (Takara Bio). TaqMan Fast Advanced Master Mix (Life Technologies) was used in a final volume of 20 μl containing Sod1, Cat, Gpx1, Gsr, Gss, Cyp11a1 and Gapdh probes designated as Rn00566938_m1, Rn00560930_m1, Rn00577994_g1, Rn01482159_m1, Rn00564188_m1, Rn00568733_m1, and Rn99999916_s1, respectively (TaqMan Gene Expression Assay; Life Technologies).