Total RNA was extracted from the total cell lysates with a PureLink RNA Mini kit (Invitrogen, San Diego, CA, USA), according to the manufacturer’s instructions. The complementary DNA was synthesized from 1 µg/mL of total RNA using SuperScript III First-Strand Synthesis System for RT-PCR (Thermo Scientific). Real-time PCR was performed using the StepOnePlus Real-Time PCR System using the TaqMan probe with the TaqMan Real-Time PCR Master Mixes (Applied Biosystems, Foster City, CA, USA). The relative mRNA expression levels were quantified using the threshold cycle (Ct) value, and the Ct value was normalized using the levels of the mouse GAPDH gene as an endogenous reference.
Taqman real time pcr master mixes
Manufactured by Thermo Fisher Scientific
Sourced in United States
TaqMan® Real-Time PCR Master Mixes are pre-formulated reagents for real-time PCR analysis. They contain all the necessary components, including DNA polymerase, dNTPs, buffer, and fluorescent probes, to enable efficient and accurate quantification of target DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Superscript 3 first strand synthesis system for rt pcr, by Thermo Fisher Scientific (1 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (1 mentions)
Cfx96 real time system, by Bio-Rad (1 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (1 mentions)
Biophotometer, by Eppendorf (1 mentions)
Nucleospin rna kit, by Macherey-Nagel (1 mentions)
Nanodrop, by Thermo Fisher Scientific (1 mentions)
Goscript reverse transcription system, by Promega (1 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Live dead kit, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by GE Healthcare (1 mentions)
Superscript first strand synthesis system for rt pcr, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by GE Healthcare (1 mentions)
Minimum essential medium (mem), by GE Healthcare (1 mentions)
Trypsin edta, by GE Healthcare (1 mentions)
Wst 1 cell proliferation assay kit, by Takara Bio (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
7 protocols using taqman real time pcr master mixes
1
RNA Extraction and Quantitative RT-PCR
2
Gene Expression Analysis by qRT-PCR
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Total RNA was extracted from cells using the RNeasy Mini Kit (Qiagen). Four micrograms of RNA was reversed transcribed into cDNA using the Superscript III First-Strand Synthesis System (Life Technologies, ThermoFisher) according to the manufacturer’s instructions. Real-time PCR was performed using TaqMan® Real-Time PCR Master Mixes (Applied Biosystems, ThermoFisher) on a CFX96 Real-Time System (Bio-Rad, Hercules, CA). Expression values were normalized using GAPDH as a reference gene. Normalization and fold-change were calculated using the ∆∆Ct method.
3
Quantifying mRNA Expression of NF-YA and FASN
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Total RNA from OS and HOB cells was extracted using TRIzol® (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Total RNA concentration was determined by measuring absorbance at a wavelength of 260 nm and purity was determined by calculating the 260/280 nm ratio with a BioPhotometer (Eppendorf, Hamburg, Germany). The mRNA expression levels of NF-YA and FASN were evaluated by RT-qPCR, using β-actin as the internal reference gene. The Two-Step RT kit (Promega Corporation, Madison, WI, USA) was used according to the manufacturer's protocol to synthesize cDNA, which was used as the template for amplification. TaqMan® Real-Time PCR Master Mixes (Thermo Fisher Scientific, Inc.) were used for amplification under the following cycling conditions: An initial denaturation step at 95°C for 1 min, followed by 40 cycles of denaturation at 95°C for 15 sec, annealing at 58°C for 20 sec and extension at 72°C for 20 sec. Data was normalized using the 2−ΔΔCq method (19 (link)). All procedures were performed according to the manufacturer's protocols, with primer sequences as listed in Table I . A total of six independent experiments were performed over multiple days.
4
Quantitative RNA Expression Analysis
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Total RNA was extracted from cells using a NucleoSpin® RNA kit (Macherey-Nagel, Düren, Germany) and was quantified by a NanoDrop (Thermo Fischer Scientific, Waltham, MA, USA). Reverse transcription was performed using a GoScript™ Reverse Transcription System (Promega, WI, USA). For qPCR analysis, TaqMan Real-Time PCR Master Mixes were used (Thermo Fischer Scientific, Waltham, MA, USA), and amplification was performed on a 7500 Real-Time PCR System. To normalize for cDNA loading, GAPDH was used as an internal control. The PCR primers used in this study are listed in Table S1 .
5
Bletilla striata Cytotoxicity Assay
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Bletilla striata (BS) was purchased from Sheng Chang Corporation (Taoyuan, Taiwan), and the WST-1 cell proliferation assay kit was obtained from Takara Bio USA, Inc. (Mountain View, CA, USA). Antibiotics, trypsin–EDTA, DMEM, MEM and FBS were purchased from GE Healthcare (Little Chalfont, UK). Live/dead kit, SuperScript first-strand synthesis system for RT-PCR, high-capacity cDNA reverse transcription kits, TaqMan real-time PCR master mixes, probes, and TRIzol were purchased from ThermoFisher Scientific (Waltham, MA, USA). All other chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA).
6
Duplex fluorescence qPCR detection specificity
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To determine detection specificity of duplex fluorescence qPCR, a 50 μL reaction volume was used, containing 1 μL (20 pmol) of each forward and reverse primer, 0.5 μL (10 pmol) of TaqMan probes, 0.5 μL (1 × 10−1 ng/μL) of standard positive plasmid, 25 μL of TaqMan™ Real-Time PCR Master Mixes (Thermo Fisher Scientific, MA, USA), and ddH2O to 50 μL. The following thermocycling program was used: 40–45 cycles of 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 30 s with an initial cycle of 95 °C for 30 s, using the Roche LightCycler 480 real time PCR system (Hoffmann-La Roche Ltd, Basel, Schweiz). All amplifications and detections were conducted using a 96-well reaction plate with optical caps (Sangon Biotech, Shanghai, China). At each cycle, accumulation of PCR products were detected by monitoring the increase in reporter dye fluorescence of the TaqMan probes. To test sensitivity, the standard positive plasmid was diluted into different concentrations (1 × 10−1 to 1 × 10−8 ng/μL) and the lowest detection efficiency and number of copies were determined.
7
Hspa8 Expression Analysis in MRL/N-1 Cells
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The mRNA of MRL/N-1 cells treated with P140 or ScP140 for indicated times and concentrations were extracted using RNeasy Mini kit (Qiagen, 75704) according to manufacturer’s instructions. The first strand cDNA was synthetized from 200 ng of RNA using ImProm-II Reverse Transcriptase and oligo(dT)15 (Promega, A3802 and C1101). Ten ng of cDNA were then amplified using Taqman Real-Time PCR Master Mixes (ThermoFisher Scientific, 10525395), followed by qPCR analysis of the cDNA amount using the StepOneTM real-time PCR system (ThermoFisher Scientific). The primers for Hspa8 (4448892) and endogenous controls, Actb (4453320), Gusb (4453320) and Hprt1 (4453320) were purchased from ThermoFisher Scientific. The data analysis was done using the correspondent StepOneTM software and the amount of Hspa8 level was normalized with the levels of the three endogenous controls.
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