Rotor gene 3000 real time dna analysis system

Total RNA was extracted using a Total RNA Isolation Kit with a mini-column system (A&A Biotechnology, Gdynia, Poland). RNA concentration and purity were determined by using NanoQuant Plate Infinite M200 PRO reader (Tecan Austria GmbH, Grodig, Austria) and the corresponding software. Total RNA (1 μg) was transcribed into cDNA using 300 ng of random primers and SuperScript II Reverse Transcriptase (Invitrogen Life Technologies, Carlsbad, CA, USA). The evaluation of the mRNA level for selected genes was performed by quantitative real-time polymerase chain reaction (qRT-PCR) by using a Rotor-Gene 3000 Real-Time DNA analysis system (Corbett Research, Morklake, Australia). cDNA was amplified with KAPA SYBR FAST qPCR Kit Universal 2× qPCR Master Mix (Kapa Biosystems, Cape Town, South Africa). We used 200 nM of each primer and 25 ng cDNA template per reaction. The annealing temperature for all transcripts was 56 °C. Primer sequences for RPS17, CXCL8, CCND1, VEGF, MMP2, and BCL2L1 are published elsewhere [27 (link),30 (link),110 (link)].

RNA isolation, cDNA synthesis and real-time PCR (qRT-PCR) experiments were performed as described previously [20] (link). Briefly, RNA was isolated and purified using total RNA isolation kit (A&A Biotechnology, Gdynia, Poland). Subsequently, 1 µg of RNA was transcribed into cDNA by SuperScript II Reverse Transcriptase (Invitrogen Life Technologies, Carlsbad, California, USA). qRT-PCR reactions were performed using KAPA SYBR FAST qPCR kit (Kapa Biosystems, Cape Town, South Africa) and the Rotor-Gene 3000 Real-Time DNA analysis system (Corbett Research, Mortlake, Australia). The annealing temperature for all genes was 56°C. To calculate the relative expression of target genes versus a reference gene (RPS17), a mathematical model including an efficiency correction for real-time PCR was used. The primers used in the study are listed in Table S1 in File S1.

RNA was isolated and purified using Total RNA Isolation kit with mini column system (A&A Biotechnology, Gdynia, Poland). The RNA concentration and purity were measured with a Tecan NanoQuant Plate reader (Tecan, Austria). cDNA was synthesized by using 300 ng of random primers and SuperScript II Reverse Transcriptase (Invitrogen Life Technologies, Carlsbad, CA, USA). The Rotor-Gene 3000 Real-Time DNA analysis system (Corbett Research, Morklake, Australia) was used to evaluate the gene expression by quantitative real-time polymerase chain reaction (qRT-PCR). The amplification was performed by using KAPA SYBR FAST qPCR Kit Universal 2X qPCR Master Mix (Kapa Biosystems, Cape Town, South Africa), 200 nM of each primer and 25 ng cDNA template per reaction. The primers used for Real-Time PCR were as following: MITF: 5′-ACC GTC TCT CAC TGG ATT GG-3′ and 5′-TAC TTG GTG GGG TTT TCG AG-3′; C-MYC: 5′-AAT GAA AAG GCC CCC AAG GTA GTT ATC C-3′ and 5′-GTC GTT TCC GCA ACA AGT CCT CTT C-3′; SOX2: 5′-GCT AGT CTC CAA GCG ACG-3′ and 5′-GCA AGA AGC CTC TCC TTG-3′. The annealing temperature for all genes was 56°C. The relative expression of target genes was calculated versus a reference gene RPS17 (with primers: 5′-AAT CTC CTG ATC CAA GGC TG-3′ and 5′-CAA GAT AGC AGG TTA TGT CAC G-3′), and a mathematical model including an efficiency correction for Real-Time PCR was used.

Total RNA Mini kit (A&A Biotechnology, Gdynia, Poland) was used to extract total RNA. The procedure was performed according to the manufacturer’s protocol. RNA concentration and purity were assessed using a microplate reader Infinite M200Pro (Tecan) at 260 nm and with a 260/280 nm ratio, respectively. Complementary DNA (cDNA) was synthesized using 1000 ng of total RNA, 300 ng of random primers, and 1 μL of SuperScript^® II Reverse Transcriptase (Invitrogen Thermo Fisher Scientific, Carlsbad, CA, USA). To assess transcript levels of target genes, a quantitative real-time polymerase chain reaction was performed using the Rotor-Gene 3000 Real-Time DNA analysis system (Corbett Research, Mortlake, VI, Australia). cDNA was amplified using 200 nM forward primer, 200 nM reverse primer, KAPA SYBR FAST qPCR Kit Universal 2X qPCR Master Mix (Sigma-Aldrich), and 25 ng of cDNA with annealing temperature 56 °C. The sequences of forward and reverse primers for NOXA and MCL-1 were shown elsewhere [45 (link),46 (link)]. To calculate the relative level of each transcript versus a reference gene RPS17 (primers: 5′-AAT CTC CTG ATC CAA GGC TG-3′ and 5′-CAA GAT AGC AGG TTA TGT CAC G-3′), a mathematical model with an efficiency correction was used [47 (link)]. (Supplementary Equation (S1))

The SK2 mRNA level in PVN was determined by means of qRT-PCR. Total cellular RNA was isolated from tissue samples via Trizol Reagent (15,596–026, Invitrogen, Carlsbad, CA, United States) as per the manufacturer’s protocol. The RNA was quantified by spectrophotometry (OD 260/280). RNA was transcribed to cDNA using M-MLV Reverse Transcriptase (D263915) and dT primers. PCR amplification was performed with Taq polymerase using 40 cycles at 94°C for 30 s, 58°C for 30 s, and 72°C for 1 min. The PCR primers for SK2 were 5′-TTG​TGG​AAG​GGG​CAT​AGG​AGA-3′ (sense) and 5′-AATGGAGCAGATGA CTGGAGA-3′ (antisense), for GAPDH were 5′-TCT​CTG​CTC​CTC​CCT​GTT​C-3′ (sense) and 5′-ACA​CCG​ACC​TTC​ACC​ATC​T-3′ (antisense), which were synthesized by Invitrogen Biotech Co. Ltd. (Shanghai, China). The qRT-PCR was performed with a Rotor-Gene 3,000 real-time DNA analysis system (Corbett Research, Sydney, Australia) with real-time SYBR Green PCR technology. The reaction mixtures contained diluted cDNA, SYBR Premix Ex Taq II (2×; DRR081), 10 μM of each gene-specific primer, and nuclease-free water in a final volume of 10 μl. The cDNA results were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) measured for the same sample.

Total RNA Isolation kit (A&A Biotechnology, Gdynia, Poland) was used to extract and purify RNA. The RNA concentration and purity were evaluated with a Tecan NanoQuant Plate reader (Tecan Group Ltd., Salzburg, Austria) at 260 nm and with 260/280 nm ratio, respectively. Total RNA (1 μg) was transcribed into cDNA using 300 ng of random primers and SuperScript II Reverse Transcriptase (Invitrogen Thermo Fisher Scientific, Carlsbad, CA, USA). Transcript levels of selected genes were assessed by quantitative real-time polymerase chain reaction (Real-Time PCR) using the Rotor-Gene 3000 Real-Time DNA analysis system (Corbett Research, Mortlake, Australia). Sequences of primers used in Real-Time PCR experiments are shown in Suppl. Table S1. Amplification was performed using KAPA SYBR FAST qPCR Kit Universal 2X qPCR Master Mix (Kapa Biosystems, Cape Town, South Africa), 200 nM of each primer, and 25 ng of cDNA per reaction. The annealing temperature for all transcripts was 56°C. To calculate the relative expression of target genes versus a reference gene RPS17, a mathematical model including an efficiency correction was used.

Total RNA was extracted and purified using the Total RNA Mini kit (A&A Biotechnology, Gdynia, Poland) according to the manufacturer’s protocol. RNA concentration was assessed using a NanoQuant Plate and a microplate reader Infinite M200Pro Tecan (Tecan) at 260 nm, and the purity of RNA samples was determined using a 260/280 nm ratio. An amount of 1 μg of total RNA was transcribed into cDNA using SuperScript II Reverse Transcriptase (Invitrogen Thermo Fisher Scientific, Carlsbad, CA, USA) and 300 ng of random primers. The quantitative real-time polymerase chain reaction was performed using a Rotor-Gene 3000 Real-Time DNA analysis system (Corbett Research, Mortlake, Australia). cDNA was amplified using a KAPA SYBR FAST qPCR Kit Universal 2X qPCR Master Mix (Sigma-Aldrich), 200 nM of each primer, and 25 ng of cDNA per reaction. The annealing temperature for all genes was 56 °C. Primer sequences were published previously for tyrosinase (TYR), ribosomal protein S17 (RPS17) [7 (link)], and IL-8 [8 (link)]. The relative abundance of each transcript was normalized to the level of RPS17 mRNA using a mathematical model with regard to the efficiency ratio.