Quantitects reverse transcription kit

The relative gene expression of caspase 3, caspase 9, Notch 1, Cyclin D1, NF-кB, IL-6, and VEGF was evaluated by qRT-PCR employing GAPDH as a housekeeping gene. The sequences of primers are listed in Table 1. The extraction of total RNA was achieved using TRIzol reagent (15,596,026) (Life Technologies, United States).
The reverse transcription process was performed by QuantiTects Reverse transcription kit (Qiagen, United States). The reaction mixtures consisted of complementary DNA amplicons, primers, and Syber green master mix (Maxima SYBR Green/qPCR Master Mix, Thermo Fisher Scientific, United States). The Livak method was employed to compute the fold change in the gene expression in comparison to the control group (calibrator) (Livak and Schmittgen, 2001 (link)).

The extracted total RNA using TRIzols (Life Technologies, Carlsbad, CA, USA) [62 (link)] was reverse-transcribed into cDNA using the QuantiTects reverse transcription kit (Qiagen, Hilden, Germany). The cDNA was amplified using the Maximas SYBR green/fluorescein qPCR master mix using the primers listed in Table S1.

The total RNA was first collected using the Trizol reagent (#15596026, Life Technologies Corporation, Carlsbad, CA, USA), then 1 μg of total RNA was reverse transcribed into cDNA by using a QuantiTects Reverse Transcription Kit (#205311, Qiagen Sciences Inc., Germantown, MD, USA). C-DNA was amplified via a Maximas SYBR Green/Fluorescein qPCR Master Mix (#K0241, Thermo Fisher Scientific Inc., Carlsbad, CA, USA) through specific primers that were prepared according to the manufacturer’s protocol (Table 1). For PCR assay, 12.5 μL Maxima SYBR Green/ Fluorescein qPCR Master Mix (2X) was mixed with 1 μL cDNA template, 0.3 μL forward primer, 0.3 μL reverse primer, and nuclease-free water to complete the volume to 25 μL. The conditions were designed as follows: 10 min at 95 °C, followed by 45 cycles of 95 °C for 10 s, 60 °C for 15 s, and 72 °C for 15 s. β-Actin was applied as an internal reference for miRNA. Rotor-Gene^® Q with software version 2.1.0 (Qiagen Sciences Inc., Germantown, MD, USA) collected data automatically and analyzed the value of the threshold cycle (Ct), which normalized to an average Ct value of the house-keeping genes (∆Ct); 2^-ΔΔCt was used for calculating relative gene expression fold [26 (link)].

For total RNA purification from testicular samples, the TRIzol reagent (Life Technologies, Inc, Carlsbad, CA, USA) was utilized. In a two-step technique RT-PCR process, 1 μg of total RNA was reverse-transcribed into single-stranded complementary DNA using the QuantiTects Reverse Transcription Kit (Qiagen, Germantown, MD, USA) and a random primer hexamer. Maximas SYBR Green/Fluorescein qPCR Master Mix was used to amplify C-DNA amplicons using particular primers produced according to the manufacturer’s procedure (Table S1).
Each sample was tested in duplicate with real-time PCR, and the mean values of the duplicates were used for further analysis. Finally, the 2^−ΔΔCT method was measured relative to mRNA expression, and then it was normalized at GAPDH [23 (link),24 (link)].

Using beta-actin as a housekeeping gene in qRT-PCR, the relative gene expression of TNF-α, VEGF, and caspase 3 was determined. Table 1 shows an assortment of primer sequences. The TRIzol reagent (15596026) from Life Technologies, Van Allen Way, Carlsbad, CA, USA, was used to extract total RNA. The QuantiTects Reverse Transcription Kit (Qiagen, Germantown, MD, USA) was used to perform the reverse transcription procedure. Primers, complementary DNA amplicons, and Syber green master mix (Maxima SYBR Green/qPCR Master Mix, Thermo Fisher Scientific, Third Avenu, Waltham, MA, USA) were included in the reaction mixtures. The fold change in gene expression concerning the calibrator control group was calculated using the Livak technique [84 (link)].