Genelute total rna miniprep kit

Total RNA from cells and liver tissues was isolated using GenElute Total RNA Miniprep Kit (Sigma) and SV total RNA isolation system (Promega Corporation, WI) respectively according to manufacturer’s instructions. The RNA concentration was quantitated by a UV spectrophotometer (NanoDrop Technologies, Wilmington, DE). Total RNA (1 μg) was reverse-transcribed using iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA). All the primers were purchased from Sigma-Genosys (Haverhill, UK). Real-time PCR was performed using 2x SensiMix SYBR and Fluorescein Kit (Bioline, QT615-05, Luckenwalde, Germany), 20 ng cDNA and pre-tested gene-specific primer sets (listed in Supplementary Tables 2 and 3). The cycling conditions for the BioRad CFX384 Real-Time PCR detection system were 95 °C for 10 min, 40 cycles of 95 °C/15 sec, 58 °C/15 sec and 72 °C/15 sec. Finally, cycle threshold (Ct) values were normalized to reference gene GAPDH and fold changes in expression were calculated using the 2^−ΔΔCt method.

1 μg of total RNA, isolated with GenElute™ total RNA Miniprep Kit (Sigma, Milan, Italy), was reverse transcribed as described previously [11 (link)]. For real time PCR (35 cycles), cDNA was amplified with GoTaq^® qPCR Master Mix (Promega, Madison, WI, USA) along with the following primers (5 pmol each): GLUL (for 5′-TCATCTTGCATCGTGTGTGTG-3′, rev 5′-CTTCAGACCATTCTCCTCCCG-3′); RPL-15 (for 5′-GCAGCCATCAGGTAAGCCAAG-3′, rev 5′-AGCGGACCCTCAGAAGAAAGC-3′); SLC38A2 (for 5′-ATGAAGAAGGCCGAAATGGGA-3′, rev 5′-TGCTTGGTGGGGTAGGAGTAG-3′); SLC1A5 (for 5′-TGGTCTCCTGGATCATGTGG-3′, rev 5′-TTTGCGGGTGAAGAGGAAGT-3′); SLC7A5 (for 5′-GTGGACTTCGGGAACTATCACC-3′, rev 5′-GAACAGGGACCCATTGACGG-3′). Quantitative PCR was performed in a 36-well Rotor Gene 3000 (Corbett Research, Rotor-Gene™ 3000, version 5.0.60, Mortlake, Australia). Each cycle consisted of a denaturation step at 95 °C for 30 s, followed by separate annealing (30 s, 55–58 °C) and extension (30 s, 72 °C) steps. Fluorescence was monitored at the end of each extension step. A no-template, no-reverse transcriptase control was included in each experiment. At the end of the amplification cycles a melting curve analysis was added. Data analysis was performed according to the Relative Standard Curve Method. [51 (link)] Expression data were normalized to RPL-15 mRNA expression.

Enzymatically isolated testicular cells and cells developed in cultures were mixed with lysis buffer and 2-mercaptoethanol mixture (10 µL 2-ME/1 mL lysis solution) (GenElute Total RNA Miniprep Kit; Sigma, St. Louis, MO, USA). Synthesis of cDNA was performed according to the qScript cDNA Synthesis Kit (Quantabio, Beverly, MA 01915, USA), using random hexamers, and qPCR was performed using specific primers for PEDF, PEDF-R and spermatogenesis markers as presented in Table 1.
The reactions were conducted following the 2 × qPCRBIO SyGreen Blue Mix Hi-ROX (PCR Biosystems Ltd., Aztec House, 397–405 Archway Road, London, UK) protocol and were performed using the LightCycler 96 real-time PCR machine (Roche, Roche Diagnostics Corporation, Roche CustomBiotech, Indianapolis, IN, USA). The PCR profile included a three-step amplification program and subsequent melting, described as follows: pre-incubation (1 cycle) 95 °C for 120 s; amplification (45 cycles) 95 °C for 10 s, 60 °C for 10 s, 72 °C for 10 s; melting (1 cycle) 95 °C for 10 s, 65 °C for 60 s, 97 °C for 1 s. The PCR products were identified and distinguished using the melting curve excepted. The relative quantity of the gene expression was analyzed using the 2−∆∆C_t method. The results were expressed as the fold of increase related to the GAPDH of the same examined sample.

Cellular RNA was isolated using a GenElute Total RNA MiniPrep kit (Sigma). Following isolation and treatment with DNase (Sigma), RNA was reverse transcribed using an iScript cDNA synthesis kit (Bio-Rad) containing 1 μg of sample RNA per reaction. RT-qPCR was conducted using IQ SYBR green SuperMix (Bio-Rad) in a Bio-Rad CFX96 Touch real-time PCR detection system as well as an Applied Biosystems StepOne Plus real-time PCR machine. A modified threshold cycle (ΔC_T) method was used to calculate gene expression using human actin for normalization. Primer sequences for actin, ZIKV, IFN-β, IFN-λ, and ISGs have been previously published (43 (link)). Additional primer sequences can be found in Table S2 in the supplemental material.

Total RNA from cells and liver tissues was isolated using GenElute Total RNA Miniprep Kit (Sigma) and SV total RNA isolation system (Promega Corporation, Madison, WI, USA), respectively, according to the manufacturer's instructions. The RNA concentration was quantified with a UV spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). Total RNA (1 μg) was reverse transcribed using the iScript cDNA Synthesis Kit (Bio‐Rad, Hercules, CA, USA). All primers were purchased from Sigma‐Genosys (Haverhill, UK). Real‐time PCR was performed using 2× SensiMix SYBR and Fluorescein Kit (Bioline, QT615‐05, Luckenwalde, Germany), 20 ng cDNA and pretested gene‐specific primer sets (listed in Appendix Table S3). The cycling conditions for the Bio‐Rad CFX384 Real‐Time PCR detection system was 95°C for 10 min, 40 cycles of 95°C/15 s, 72°C/15 s, and 58°C/15 s. Finally, cycle threshold (Ct) values were normalized to reference gene GAPDH or 18S and fold changes in expression were calculated using the 2^−ΔΔCt method.

Total RNA from cells and liver tissues was isolated using GenElute Total RNA Miniprep Kit (Sigma) and SV total RNA isolation system (Promega Corporation) respectively according to manufacturer's instructions. The RNA concentration was quantitated by a UV spectrophotometer (NanoDrop Technologies). Total RNA (1 μg) was reverse transcribed using iScript cDNA Synthesis Kit (Bio‐Rad). All the primers were purchased from Sigma‐Genosys. Real‐time PCR was performed using 2× SensiMix SYBR and Fluorescein Kit (Bioline, QT615‐05), 20 ng cDNA and pre‐tested gene‐specific primer sets (Tables S3 and S4). The cycling conditions for the BioRad CFX384 Real‐Time PCR detection system were 95°C for 10 minutes, 40 cycles of 95°C/15 sec, 58°C/15 sec, and 72°C/15 sec. Finally, cycle threshold (Ct) values were normalized to reference gene GAPDH and fold changes in expression were calculated using the 2^−ΔΔCt method.