The expression levels of miR-23a-3p, miR-101-3p and miR-let-7c in plasma samples of all studied subjects were evaluated by qPCR procedure. The qPCR assays were performed in triplicate for all specimens using Syber Green-I dye in AccuPower® 2X GreenStarTM qPCR Master Mix (Bioneer Inc., Seoul, South Korea) and miRNA-speci c primers according to the manufacturer's protocol, on the StepOnePlus™ Real-Time PCR System (Applied Biosystems, Foster City, USA). In a total volume of 25µl, 100 ng cDNA was mixed with 12.5 µL of 2X SYBR Green PCR master mix (Bioneer Inc., Seoul, South Korea) and 10 pmol of each primer pairs for miR-23a-3p, miR-101-3p and miR-let-7c and housekeeping gene U6 small nuclear RNA (snRNA). Thermal cycling program consisted of initial denaturation step at 95°C for 30 s, followed by 40 cycles at 94°C for 5 s, 60°C for 1 min. In order to normalize the variability of miR-23a-3p, miR-101-3p and miR-let-7c expressions, we evaluated the expression level of the housekeeping gene U6 snRNA, as an internal control. The no template control (NTC) was also used as a negative control to monitor contamination. The relative changes in expression levels of miR-23a-3p, miR-101-3p and miR-let-7c were calculated using the comparative 2 -ΔΔCT method.
Sybr green pcr master mix
Manufactured by Bioneer
SYBR Green PCR Master Mix is a ready-to-use solution for real-time quantitative PCR (qPCR) applications. It contains all the necessary components, including SYBR Green I dye, DNA polymerase, dNTPs, and buffer, to perform sensitive and efficient real-time PCR reactions.
Automatically generated - may contain errors
Lab products found in correlation
Accupower 2x greenstar qpcr master mix, by Bioneer (2 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Anti mouse cd16 cd32, by BD (1 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (1 mentions)
Gm csf, by Thermo Fisher Scientific (1 mentions)
Recombinant human rankl, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated mouse and rabbit igg antibodies, by BD (1 mentions)
Ifn γ, by Thermo Fisher Scientific (1 mentions)
Murine m csf, by Thermo Fisher Scientific (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
Oligonucleotides, by Bioneer (1 mentions)
Avian myeloblastosis virus reverse transcriptase, by Promega (1 mentions)
Applied biosystems 7300 fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Accupower cyclescript rt premix dn6 kit, by Bioneer (1 mentions)
Cinnapure rna extraction kit, by Sinaclon (1 mentions)
6 protocols using sybr green pcr master mix
1
Plasma miRNA Expression Profiling
2
Plasma miRNA Expression Profiling
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The expression levels of miR-23a-3p, miR-101-3p and miR-let-7c in plasma samples of all studied subjects were evaluated by qPCR procedure. The qPCR assays were performed in triplicate for all specimens using Syber Green-I dye in AccuPower® 2X GreenStarTM qPCR Master Mix (Bioneer Inc., Seoul, South Korea) and miRNA-speci c primers according to the manufacturer's protocol, on the StepOnePlus™ Real-Time PCR System (Applied Biosystems, Foster City, USA). In a total volume of 25µl, 100 ng cDNA was mixed with 12.5 µL of 2X SYBR Green PCR master mix (Bioneer Inc., Seoul, South Korea) and 10 pmol of each primer pairs for miR-23a-3p, miR-101-3p and miR-let-7c and housekeeping gene U6 small nuclear RNA (snRNA). Thermal cycling program consisted of initial denaturation step at 95°C for 30 s, followed by 40 cycles at 94°C for 5 s, 60°C for 1 min. In order to normalize the variability of miR-23a-3p, miR-101-3p and miR-let-7c expressions, we evaluated the expression level of the housekeeping gene U6 snRNA, as an internal control. The no template control (NTC) was also used as a negative control to monitor contamination. The relative changes in expression levels of miR-23a-3p, miR-101-3p and miR-let-7c were calculated using the comparative 2 -ΔΔCT method.
3
Osteoclastogenesis Induction Protocol
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Human recombinant RANKL, murine M-CSF (also called CSF-1), IFN-γ, IL-4, GM-CSF, and TNF-α were procured from PeproTech (London, UK). Minimum essential medium (α-MEM), phosphate buffered saline (PBS), penicillin, and streptomycin were purchased from HyClone (Logan, UT, USA). Oligonucleotides and SYBR Green PCR Master Mix were bought from Bioneer (Daejeon, Korea) and Toyobo (Osaka, Japan), respectively. Antibodies against the following antigens were used in this study: HO-1 (Enzo Life Sciences, Farmingdale, NY, USA), Nrf2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), peroxiredoxin 1 (Prx1) (AbFrontier, Seoul, Korea), and anti-mouse CD16/CD32 (BD Bioscience, San Jose, CA, USA). Horseradish peroxidase-conjugated mouse- and rabbit-IgG antibodies were purchased from BD Pharmingen (San Diego, CA, USA). Other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated.
4
Cardiac Muscle Total RNA Extraction and qPCR
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Total RNA was extracted from the cardiac muscle tissue using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), following the manufacturer's protocol. cDNA was generated using avian myeloblastosis virus reverse transcriptase (Promega Corporation, Madison, WI, USA) and oligo (dT) primers according to the manufacturer's instructions. Primer sequences and reaction conditions are listed in Table I . Real-time PCR was performed in an Applied Biosystems 7300 Fast Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) with SYBR Green PCR Master Mix (Bioneer Corporation, Daejeon, Korea). Primer sequences used and thermocycling conditions are presented in Table I . β-actin mRNA amplified from the same samples served as an internal control. The relative β-actin expression of each targeted gene was normalized by subtracting the corresponding threshold cycle (Ct) values using the 2−ΔΔCq comparative method (14 (link)).
5
HLA-G Isoform Expression in Vaginal Discharge
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Total RNA was isolated from vaginal discharge using CinnaPure RNA extraction kit (SinaClon BioScience Co., Tehran, Iran), according to the manufacturer's procedure. Total RNA was reversely transcribed using the AccuPower CycleScript RT PreMix (dN 6) kit (Bioneer Inc., Seoul, South Korea). qPCR was performed using specific primers for HLA-G1, HLA-G5 isoforms and elongation factor 1 (reference gene) genes (Table 2 ) with SYBR Green qPCR Master Mix.
Quantitative PCR reactions were done in a 20 μL volume including 1x SYBR Green PCR master mix (Bioneer Inc., Seoul, South Korea), 1 μL of forward and reverse primers, and 2 μL of cDNA following the manufacturer's instructions. After a primary 10 min at 95°C as activation step, 40 cycles comprising of denaturation at 95°C, 30 s, annealing at 59°C, 30 s, and extension at 72°C, 45 s were performed by BioRad iQ5 Multicolor Real-Time PCR (Bio-Rad Laboratories, Hercules, CA, USA) detection system. Gene expression was analyzed by comparing with the reference gene in each PCR run.
Quantitative PCR reactions were done in a 20 μL volume including 1x SYBR Green PCR master mix (Bioneer Inc., Seoul, South Korea), 1 μL of forward and reverse primers, and 2 μL of cDNA following the manufacturer's instructions. After a primary 10 min at 95°C as activation step, 40 cycles comprising of denaturation at 95°C, 30 s, annealing at 59°C, 30 s, and extension at 72°C, 45 s were performed by BioRad iQ5 Multicolor Real-Time PCR (Bio-Rad Laboratories, Hercules, CA, USA) detection system. Gene expression was analyzed by comparing with the reference gene in each PCR run.
6
Quantitative RT-PCR of Insect Genes
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The RT-qPCR analysis was conducted using QuantStudio5 (Thermo Fisher Logical, made in Singapore). The SYBR Green PCR Master Mix (BiONEER, Daejeon, Republic of Korea) was employed in a triplicate reaction system following the manufacturer’s instructions. Primers were used at a concentration of 200 nM in each reaction, with a final volume of 9 μL. The PCR protocol comprised initial denaturation at 95 °C for 1 min, followed by 40 cycles of 95 °C for 15 s, 55 °C for 15 s, and 72 °C for 30 s. A final melt-curve step confirmed the absence of non-specific amplification. Cycle threshold (Ct) values of seven candidate reference genes and the target genes MFE, Kr-h1, and Vg were obtained at the same fluorescence threshold (0.1).
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