Brain tissues or cells were sonicated and lysed with TRIzol (Invitrogen, 10296010). Total RNA was extracted according to the manufacturer's instructions and treated with DNase I (Sigma-Aldrich, AMPD1). Next, cDNA was synthesized using a high-capacity cDNA reverse transcription kit (Takara, RR037Q). The qRT-PCR analysis was performed on a Step-one System (Bio-Rad) with TB Green Mastermix (Takara, R075A). Relative mRNA expression was determined through the 2–ΔΔCt method and normalized to GAPDH expression. The primer sequences are shown in Supplementary Table 1 .
Step one system
Manufactured by Bio-Rad
Sourced in United States
The Step-one System is a thermal cycler designed for real-time PCR and reverse transcription PCR (RT-PCR) applications. It provides precise temperature control and monitoring for accurate and reproducible results.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Sybr green, by Thermo Fisher Scientific (2 mentions)
Sybr green master mix, by Takara Bio (2 mentions)
Tb green master mix, by Takara Bio (1 mentions)
High capacity cdna reverse transcription kit, by Takara Bio (1 mentions)
Dnase 1, by Merck Group (1 mentions)
M mlv, by Takara Bio (1 mentions)
5 protocols using step one system
1
Analyzing Gene Expression in Brain Tissues
2
Quantifying PVT1 and miR-128-3p in Breast Cancer
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RNA was extracted using TRIzol reagent (Invitrogen, USA). cDNA was quantified by SYBR green (Applied BioSystems, USA) on the StepOne System (Bio-Rad, USA). U6 was used as the endogenous control for miR-128-3p and GAPDH for PVT1. Each reaction was performed in triplicates and relative expression of target genes was calculated by the 2–△△Ct method for gene expression in BC cells and the 2–△Ct method for that in BC plasma. The following primers were used: PVT1 sense (5’-TGAGAACTGTCCTTA CGTGACC-3’) and antisense (5’-AGAGCACCAAGACTGGCTCT-3’); GAPDH sense (5’-GGGAGCCAAAAGGGTCAT-3’) and antisense (5’-GAGTCCTTCC ACGATACCAA-3’).
3
Extraction and qRT-PCR Analysis of RNA
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Cultured cells were lysed by TRIzol (Invitrogen), and RNA was extracted according to the manufacturer’s instruction. One microgram of total RNA from each sample was reverse transcribed using M-MLV (Takara) in a final volume of 30 μL. The polymerase chain reaction (PCR) amplification was carried out using the Step-one System (Bio-Rad) with SYBR Green Mastermix (Takara). All quantitative real-time PCR (qRT-PCR) results were carried out in duplicate and normalized to GAPDH.
4
qRT-PCR Gene Expression Analysis
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Cultured cells were lysed with TRIzol reagent (Invitrogen, USA), and RNA was extracted according to the manufacturer's instructions. One microgram of total RNA from each sample was reverse-transcribed using Moloney Murine Leukemia Virus (M-MLV; Takara, Japan) in a final volume of 20 µL. PCR amplification was carried out using a Step-one system (Bio-Rad, USA) with SYBR Green master mix (Takara, Japan). All qRT-PCR analyses were carried out in duplicate and normalized to GAPDH. The primers for the related genes are listed in table 1. GAPDH mRNA was used as an internal control.
5
Quantifying PVT1 and miR-128-3p in Breast Cancer
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RNA was extracted using TRIzol reagent (Invitrogen, USA). cDNA was quantified by SYBR green (Applied Bio-Systems, USA) on the StepOne System (Bio-Rad, USA). U6 was used as the endogenous control for miR-128-3p and GAPDH for PVT1. Each reaction was performed in triplicates and relative expression of target genes was calculated by the 2 -△△Ct method for gene expression in BC cells and the 2 -△Ct method for that in BC plasma. The following primers were used: PVT1 sense (5'-TGA GAA CTG TCC TTA CGT GAC C-3') and antisense (5'-AGA GCA CCA AGA CTG GCT CT-3'); GAPDH sense (5'-GGG AGC CAA AAG GGT CAT -3') and antisense (5'-GAG TCC TTCC ACG ATA CCAA-3').
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