According to the manufacturer’s instructions, Trisure Reagent (Bioline) was used for the extraction of total RNA. To assess the quality of RNA, the ratio A260/A280 obtained in a NanoDrop ND-1000 Spectrophotometer (Thermo Scientific, Madrid, Spain) was used. The first step in this process was to subject 1 µg RNA to reverse transcription (iScript, Bio-Rad, Madrid, Spain); 20 ng of the obtained cDNA was used as a template for real-time PCR amplifications. Using a CFX96 system (Bio-Rad), mRNA levels were analyzed for specific genes. In every PCR reaction, the cDNA template was mixed with a Brilliant SYBR green QPCR Supermix (Bio-Rad) in which glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the housekeeping gene or the primer pairs for either gene were present (Table 1 ). PCR amplifications were performed in triplicate, and to quantify the relative levels of mRNA expression for every gene tested, the average threshold cycle (Ct) numbers of triplicates were used. With the standard 2−(ΔΔCt) method, the proportion of change in mRNA expression in candidate genes was quantified. Data were expressed as percentages adjusted to controls and normalized to endogenous reference gene content [37 (link)].
Brilliant sybr green qpcr supermix
Manufactured by Bio-Rad
Sourced in United States
Brilliant SYBR green QPCR Supermix is a ready-to-use solution for quantitative PCR (qPCR) analysis. It contains SYBR Green I dye, a DNA-binding fluorescent dye, and all the necessary components for qPCR, including a DNA polymerase, dNTPs, and buffer.
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15 protocols using brilliant sybr green qpcr supermix
1
RNA Extraction and qRT-PCR Analysis
2
RNA Extraction and Quantification for Gene Expression Analysis
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Total RNA was extracted by using TRIsure Reagent (Bioline, London, UK), as instructed by the manufacturer. A260/A280 ratio in a NanoDrop ND-1000 Spectrophotometer (Thermo Scientific) was used to determinate RNA quality. Momentarily, RNA (1 µg) was subjected to reverse transcription (iScript, Bio-Rad, Madrid, Spain). An amount of 10 ng of the resulting cDNA was used as a template for real-time PCR amplifications. The mRNA levels for specific genes were determined in a CFX96 system (Bio-Rad). For each PCR reaction, cDNA template was added to Brilliant SYBR green QPCR Supermix (Bio-Rad) containing the primer pairs for either gene or for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and hypoxanthine phosphoribosyltransferase 1 (HPRT) as housekeeping genes (Supplementary Materials Table S1 ). All amplification reactions were performed in triplicate, and average threshold cycle (Ct) numbers of the triplicates were used to calculate the relative mRNA expression of candidate genes. The magnitude of change of mRNA expression for candidate genes was calculated by using the standard 2−(ΔΔCt) method. All data were normalized to endogenous reference (GAPDH and HPRT) gene content and expressed as percentage of controls.
3
Quantitative Real-Time PCR Analysis
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Total RNA was extracted by using Trisure Reagent (Bioline). RNA quality was assessed by the A260/A280 ratio in a NanoDrop ND-1000 Spectrophotometer (Thermo Scientific). RNA (250 ng) was subjected to reverse transcription (iScript, BioRad). An amount of 20 ng of the resulting cDNA was used as a template for real-time PCR amplifications. The mRNA levels for specific genes were determined in a CFX96 system (BioRad). For each PCR reaction, a cDNA template was added to a Brilliant SYBR green QPCR Supermix (BioRad) containing the primer pairs for the corresponding gene. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and hypoxanthine phosphoribosyl transferase (HPRT) were used as housekeeping genes. The sequence and information for the primers are shown in Cardeno et al. (2015) [44 (link)]. All amplification reactions were performed in triplicate and average threshold cycle (Ct) numbers of the triplicates were used to calculate the relative mRNA expression of candidate genes. The magnitude of change of mRNA expression for candidate genes was calculated by using the standard 2−(ΔΔCt) method. All data were normalized to the content of housekeeping genes and expressed as percentage of control.
4
Quantitative Gene Expression Analysis
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Total RNA was also obtained from cells using Trisure Reagent (Bioline GmbH, Berlin, Germany). RNA quality was evaluated with an A260/A280 ratio in a NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE). RNA (1 µg) was subjected to reverse transcription (iScript, BioRad, Madrid, Spain) following the manufacturer’s indications. Ten ng of the resulting cDNA was employed as a template for real-time polymerase chain reaction (PCR) amplifications. The mRNA levels for individual genes were quantified using real-time PCR in a CFX96 system (BioRad). For each PCR reaction, cDNA template was added to Brilliant SYBR green QPCR Supermix (BioRad, CA, USA) containing the primer pairs for either gene or for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and hypoxanthine phosphoribosyltransferase (HPRT) as housekeeping genes (Table 1 ). The average threshold cycle (Ct) values of all the triplicate amplification reactions were used to determine the relative mRNA expression of the candidate genes. Using the conventional 2-(ΔΔCt) technique, the magnitude of change in mRNA expression for candidate genes was determined. All data were reported as a percentage of controls after being adjusted to the content of the endogenous reference genes (GAPDH and HPRT).
5
Real-time PCR quantification of gene expression
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Total RNA was extracted by using Trisure Reagent (Bioline). RNA quality was assessed by A260/A280 ratio in a NanoDrop ND-1000 Spectrophotometer (Thermo Scientific). Briefly, RNA (1 µg) was subjected to reverse transcription (iScript, Bio-Rad, Madrid, Spain). An amount of 10 ng of the resulting cDNA was used as a template for real-time PCR amplifications. The mRNA levels for specific genes were determined in a CFX96 system (Bio-Rad). For each PCR reaction, cDNA template was added to Brilliant SYBR green QPCR Supermix (Bio-Rad) containing the primer pairs for either gene or for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and hypoxanthine phosphoribosyltransferase (HPRT) as housekeeping genes (Supplementary Materials Table S2 ). All amplification reactions were performed in triplicate and average threshold cycle (Ct) numbers of the triplicates were used to calculate the relative mRNA expression of candidate genes. The magnitude of change of mRNA expression for candidate genes was calculated by using the standard 2−(ΔΔCt) method. All data were normalized to endogenous reference (GAPDH and HPRT) gene content and expressed as relative fold-change of control.
6
Quantifying Gene Expression via qPCR
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Total RNA was extracted by using Trisure Reagent (Bioline, London, UK). RNA quality was assessed by A 260 /A 280 ratio in a NanoDrop ND-1000 Spectrophotometer (Thermo Scientific, Greenville, SC, USA). Briefly, RNA (250 ng) was subjected to reverse transcription (iScript, Bio-Rad, Madrid, Spain). An amount of 20 ng of the resulting cDNA was used as a template for real-time PCR amplifications. The mRNA levels for specific genes were determined in a CFX96 system (Bio-Rad). For each PCR reaction, cDNA template was added to Brilliant SYBR green QPCR Super-mix (Bio-Rad) containing the primer pairs for either gene or for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a housekeeping gene (Table 1). All amplification reactions were performed in triplicate and average threshold cycle (Ct) numbers of the triplicates were used to calculate the relative mRNA expression of candidate genes. The magnitude of change of mRNA expression for candidate genes was calculated by using the standard 2 -(ΔΔCt) method. All data were normalized to endogenous reference (GAPDH) gene content and expressed as relative fold-change of control.
7
Hepatic Gene Expression Analysis by RT-qPCR
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RNA from hepatic tissues was isolated to quantify gene expression by RT-qPCR. Total RNA was extracted by using TRIsure Reagent (Bioline). RNA quality was assessed by the A 260 /A 280 ratio using a NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific, Madrid, Spain). Briefly, RNA (250 ng) was subjected to reverse transcription (iScript, Bio-Rad).
An amount of 40 ng of the resulting cDNA was used as a template for RT-qPCR amplifications. The mRNA levels for specific genes were determined in a CFX96 system (Bio-Rad). For each PCR reaction, a cDNA template was added to Brilliant SYBR green QPCR Supermix (Bio-Rad) containing the primer pairs for either gene or hypoxanthine phosphoribosyltransferase (HPRT) as a housekeeping gene. All amplification reactions were performed in triplicate and the average threshold cycle (Ct) numbers of the triplicates were used to calculate the relative mRNA expression of the candidate genes. The magnitude of the change in the mRNA expression for candidate genes was calculated by using the standard 2 -(ΔΔCt) method. All data were normalized to endogenous reference (HPRT) gene content and expressed as relative fold-change of control. The sequences of the designed oligonucleotides are shown in Table S1. †
An amount of 40 ng of the resulting cDNA was used as a template for RT-qPCR amplifications. The mRNA levels for specific genes were determined in a CFX96 system (Bio-Rad). For each PCR reaction, a cDNA template was added to Brilliant SYBR green QPCR Supermix (Bio-Rad) containing the primer pairs for either gene or hypoxanthine phosphoribosyltransferase (HPRT) as a housekeeping gene. All amplification reactions were performed in triplicate and the average threshold cycle (Ct) numbers of the triplicates were used to calculate the relative mRNA expression of the candidate genes. The magnitude of the change in the mRNA expression for candidate genes was calculated by using the standard 2 -(ΔΔCt) method. All data were normalized to endogenous reference (HPRT) gene content and expressed as relative fold-change of control. The sequences of the designed oligonucleotides are shown in Table S1. †
8
Quantitative RT-PCR Gene Expression Analysis
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RNA was totally extracted by using Trisure Reagent (Bioline GmbH, Luckenwakde, Germany), following the manufacturer’s instructions. The measure of A260/A280 ratio in a NanoDrop ND-1000 Spectrophotometer (Thermo Scientific, Madrid, Spain) was used to determinate the RNA grade. Then, the reverse transcription (iScript, Bio-Rad, Madrid, Spain) was performed to obtain cDNA from RNA (1 µg). The resulting cDNA (10 ng) was used as a template for real-time PCR amplifications. The mRNA levels for specific genes were determined in a CFX96 system (Bio-Rad). For each PCR reaction, a cDNA template was added to Brilliant SYBR green QPCR Supermix (Bio-Rad, Hercules, CA, USA) containing the primer pairs for either genes or for housekeeping genes such as glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Table S3 ). All amplification reactions were performed at least thrice and average threshold cycle (Ct) numbers of these triplicates were used to obtain the relative mRNA expression of candidate genes. The magnitude of change of mRNA expression for candidate genes was calculated by using the mathematical method of 2−(ΔΔCt). Briefly, all candidate genes were normalized to housekeeping genes (GAPDH) and expresed as the control’s percentage.
9
Quantifying Gene Expression by qRT-PCR
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Total RNA was extracted by using Trisure Reagent (Bioline), as instructed by the manufacturer. RNA quality was assessed by A260/A280 ratio in a NanoDrop ND-1000 Spectrophotometer (Thermo Scientific, Madrid, Spain).
Briefly, RNA (1 µg) was subjected to reverse transcription (iScript, Bio-Rad, Madrid, Spain). An amount of 20 ng of the resulting cDNA was used as a template for real-time PCR amplifications. The mRNA levels for specific genes were determined in a CFX96 system (Bio-Rad). For each PCR reaction, cDNA template was added to Brilliant SYBR green QPCR Supermix (Bio-Rad) containing the primer pairs for either gene or for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as housekeeping genes (Table 2). All amplification reactions were performed in triplicate and average threshold cycle (Ct) numbers of the triplicates were used to calculate the relative mRNA expression of candidate genes. The magnitude of change of mRNA expression for candidate genes was calculated by using the standard 2 -(∆∆Ct) method. 19 All data were normalized to endogenous reference (GAPDH) gene content and expressed as percentage of controls.
Briefly, RNA (1 µg) was subjected to reverse transcription (iScript, Bio-Rad, Madrid, Spain). An amount of 20 ng of the resulting cDNA was used as a template for real-time PCR amplifications. The mRNA levels for specific genes were determined in a CFX96 system (Bio-Rad). For each PCR reaction, cDNA template was added to Brilliant SYBR green QPCR Supermix (Bio-Rad) containing the primer pairs for either gene or for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as housekeeping genes (Table 2). All amplification reactions were performed in triplicate and average threshold cycle (Ct) numbers of the triplicates were used to calculate the relative mRNA expression of candidate genes. The magnitude of change of mRNA expression for candidate genes was calculated by using the standard 2 -(∆∆Ct) method. 19 All data were normalized to endogenous reference (GAPDH) gene content and expressed as percentage of controls.
10
RNA Extraction and qRT-PCR Analysis
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Total RNA was extracted from cells by using Trisure Reagent (Bioline), as instructed by the manufacturer. RNA quality was assessed by A 260 /A 280 ratio in a NanoDrop ND-1000 Spectrophotometer (Thermo Scientific). RNA (1 µg) was subjected to reverse transcription (iScript, Bio-Rad, CA, USA) according to the manufacturers' protocol. An amount of 20 ng of the resulting cDNA was used as a template for real-time PCR amplifications. The mRNA levels for specific genes were determined in a MX3000P system (Stratagene). For each PCR reaction, cDNA template was added to Brilliant SYBR green QPCR Supermix (Bio-Rad) containing the primer pairs for either gene or for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and hypoxanthine phosphoribosyltransferase (β-actine) as housekeeping genes (Table 1). All amplification reactions were performed in triplicate and average threshold cycle (Ct) numbers of the triplicates were used to calculate the relative mRNA expression of candidate genes. The magnitude of change of mRNA expression for candidate genes was calculated by using the standard 2 -(ΔΔCt) method. All data were normalized to endogenous reference (GAPDH and β-actine) gene content and expressed as percentage of controls.
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