Total RNA was extracted from the H9c2 cells using the TRIzol reagent (Invitrogen) and reverse transcribed using the TaqMan microRNA reverse transcription kit or a high-capacity cDNA reverse transcription kit (Thermo Fisher). The quantitative real-time polymerase chain reaction (qRT-PCR) was performed at 95 °C for 5 min followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min using the SYBR master mix kit (Takara, Otsu, Japan). The relative expressions of miR-144-3p and TUG1 were analyzed with U6 or GAPDH as an internal control using the 2−ΔΔCt method.20 (link) The special primers are listed as follows: TUG1 forward primer: 5′-CTGAAGAAAGGCAACATC-3′, reverse primer: 5′-GTAGGCTACTACAGGATTTG-3′; miR-144-3p forward primer: 5′-GCGCGCTACAGTATAGATGATG-3′, reverse primer: 5′-GCGCGCTACAGTATAGATGATG-3′; U6 forward primer: 5′-GGAGCGAGATCCCTCCAAAAT-3′, reverse primer: 5′-GGCTGTTGTCATACTTCTCATGG-3′; GAPDH forward primer: 5′-AACGACCCCTTCATTGACCTC-3′, reverse primer: 5′-CCTTGACTGTGCCGTTGAACT-3′.
Sybr master mix kit
Manufactured by Takara Bio
Sourced in Japan
The SYBR Master Mix Kit is a reagent kit designed for real-time quantitative PCR (qPCR) analysis. It contains a pre-mixed solution of SYBR Green I dye, DNA polymerase, necessary buffers, and other essential components required for qPCR reactions.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (9 mentions)
Steponeplus system, by Takara Bio (2 mentions)
Lightcycler 480, by Roche (2 mentions)
Cdna synthesis kit, by Takara Bio (2 mentions)
Lightcycler 96 instrument, by Roche (2 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Reliance select cdna synthesis kit, by Bio-Rad (1 mentions)
Superscript 2 rt pcr, by Thermo Fisher Scientific (1 mentions)
Specific primers, by Genechem (1 mentions)
Mx3000p qpcr system, by Agilent Technologies (1 mentions)
Trizol reagent, by Sangon (1 mentions)
Sybr premix ex taq 2, by Takara Bio (1 mentions)
Reverse transcription kit, by Takara Bio (1 mentions)
Real time pcr detection system, by Bio-Rad (1 mentions)
Lightcycler 480 real time pcr system, by Roche (1 mentions)
M mlv reverse transcription kit, by Promega (1 mentions)
Lightcycler 480 2, by Roche (1 mentions)
12 protocols using sybr master mix kit
1
Quantitative Analysis of miR-144-3p and TUG1 Expression
2
Quantitative Analysis of p21 Gene Expression
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The total RNA was isolated from the treated cardiomyocytes using the Trizol reagents (Thermo, Massachusetts, USA). The extracted RNA was treated with DNase I (Thermo, Massachusetts, USA) for 10 min to remove contaminated cDNA. The quality and concentration of RNA were validated using a Nanodrop 2000 spectrophotometer (Thermo, Massachusetts, USA). The wavelength absorption ratio (260/280 nm) was between 1.8 and 2.0 for all preparations. A total 1 µg of RNA was transcribed into cDNA utilizing the Reliance Select cDNA Synthesis Kit (Bio-Rad, California, USA). In the present study, the PCR reaction was conducted utilizing the SYBR Master Mix kit with a 25-μL reaction system and the StepOne-Plus system (Takara, Tokyo, Japan) according to the following procedure: denaturing at 95 °C for 30 s, annealing at 60 °C for 1 min and extending at 95 °C for 5 s for 40 cycles, and 72 °C 10 min, 1 cycle. Finally, the 2−ΔΔCt method was used to determine the relative expression level of target genes with β-actin used for normalization. The following primers were used in this study: p21, (F: 5′ -TGTTCCACACAGGAGCAAAG-3′, R: 5′-AACACGCTCCCAGACGTAGT-3′), β-actin (F: 5′-CTGCCCTGGCTCCTAGCAC-3′, R: 5′- CGGACGCAGCTCAGTAACAGTCCG-3′).
3
RNA Extraction and Quantitative RT-PCR
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Total RNA was separated from dissected tumor and non-tumor tissues, and three cell lines, using the TRIzol® reagent (Sangon Biotech Co., Ltd.), based on the manufacturer's protocols. RT was executed using 2 µg total RNA, 1 µl (200 units) RT-PCR SuperScript II (Invitrogen; Thermo Fisher Scientific, Inc.) and 50 µM decamer at 37°C for 50 min. Specific primers targeting OIP5 were designed by Shanghai GeneChem Co., Ltd. as follows: 5′-TGGCATTGAAGGTTCACTCA-3′ (forward) and 5′-AGGGCAGCATGGGTAGAATA-3′ (reverse), with a product length of 189 bp. RT-qPCR was performed by SYBR® Master Mix Kit (Takara Biotechnology Co., Ltd., Dalian, China) on a Mx3000P qPCR system (Agilent Technologies, Inc., Santa Clara, CA, USA) and LightCycler 480 (Roche Diagnostics, Indianapolis, IN, USA). The housekeeping gene GAPDH was used for normalization. Data were calculated using the Pfaffl method (22 (link)). PCR primers used for validating the microarray data are listed in Table II . The thermal profile conditions were 30 sec at 95°C, 30 sec at 62°C and 30 sec at 72°C for 40 cycles, and a final extension at 72°C for 5 min.
4
RNA Extraction and qRT-PCR Analysis
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TRIzol (Shanghai Pufei Biotech Co., Ltd., Shanghai, China) was used to extract and purify total RNA from cells according to the manufacturer’s instructions. Gene expression levels were determined using the SYBR Master Mix Kit (TAKARA, Kusatsu, Japan) on a LightCycler480 qRT-PCR platform (Roche, Basel, Switzerland). The following cycling conditions were used: 95°C for 30 s; 40 cycles of 95°C for 5 s and 60°C for 30 s. mRNA expression levels were calculated using the 2−ΔΔCT method. GAPDH was used as the internal standard.
5
Inflammatory Cytokine Expression in Macrophages
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The mRNA expression levels of IL-1β, IL-6, and TNF-α were determined using reverse transcription-polymerase chain reaction (RT-PCR). RAW264.7 cells were attached to a 6-well microplate and treated with different doses (0.2, 2, and 20 μg/ml) of BM2U, with or without LPS (1 μg/ml), for 16 h. Total RNA was extracted from RAW264.7 macrophages using the TRIzol reagent from Invitrogen (USA), and processed using a cDNA Synthesis kit from TAKARA (Japan). SYBR master mix kit from TAKARA was used for RT-PCR, and cDNA was amplified using specific primers (Table 1 ). Quantitative real-time RT-PCR reactions were performed on a Light Cycler 96 Instrument from Roche (Basel, Switzerland). Relative quantitative evaluation of each gene was performed by the comparative cycle threshold method [20 (link)].
6
Quantification of Syt-7 Gene Expression
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Total RNA was extracted using Trizol reagent (Pufei Biotech Co, Ltd, Shanghai, People’s Republic of China) according to the manufacturer’s instructions. Synthesis of cDNA was performed by reverse transcription using Reverse Transcription System reagents (Promega Corporation, Fitchburg, WI, USA). The expression of Syt-7 was quantified by RT-qPCR analysis using SYBR Premix Ex Taq II (Takara, Kyoto, Japan) with GAPDH as an internal reference. The following sequences of primers were used: 5′-TGACTTCAACAGCGACACCCA-3′ (upstream) and 5′-CACCCTGTTGCTGTAGCCAAA-3′ (downstream) for SYT-7 and 5′-ACTCCATCATCGTGAACATCATC-3′ (upstream) and 5′-TCGAAGGCGAAGGACTCATTG-3′ (downstream). Quantitative PCR was performed according to Takara SYBR Master Mix kit instructions: 95°C for 15 s, followed by 45 cycles of 95°C for 5 s and 60°C for 20 s. The relative gene expression levels were calculated and statistically compared using the 2−ΔΔCT analysis program.13 (link)
7
Total RNA Extraction and qRT-PCR Analysis
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TRIzol (Shanghai Pufei Biotech Co., Ltd) was used to extract and purify the total RNA from the tissues according to the manufacturer’s instructions, and cDNA was synthesized using the Reverse Transcription Kit (TAKARA). Gene expression was detected using the SYBR Master Mix Kit (TAKARA) in Real-Time PCR platform LightCycler480 (Roche), and the reaction conditions were: 95°C for 30s; 40 cycles of 95°C for 5s and 60°C for 30s; and dissociation at 95°C for 15s, 60°C for 30 s, and 95°C for 15s. The relative gene expression levels were calculated using the 2−ΔΔCt method [29 (link)]. GAPDH was used as the internal reference.
8
Quantitative RT-PCR Analysis of miRNA and mRNA
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Total RNA was extracted using TRIzol reagent (Invitrogen) and reverse transcribed using TaqMan microRNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). The qRT-PCR was performed with SYBR master mix kit (Takara, Otsu, Japan) on Real-Time PCR Detection System (Bio-Rad) following the procedure: 95 °C for 5 min, followed by 35 cycles of 95 °C for 15 s and 60 °C for 1 min. The relative expressions of targeted RNAs were analyzed with U6 or GAPDH as internal control using 2−ΔΔCt method.14 (link) The special primers were listed as follows: miR-107 forward primer: 5′-GTTAAGTCAGAGCGGGGCTT-3′, reverse primer: 5′-CACTCCGCTTTTTCAGTGCC-3′; U6 forward primer: 5′-CTCGCTTCGGCAGCACA-3′, reverse primer: 5′-AACGCTTCACGAATTTGCGT-3′; ZNF217 forward primer: 5′-GTTGTTCCATTCCGAGCTACA-3′, reverse primer: 5′-GGTAGGCCGGTGTTGCATTA-3′; GAPDH forward primer: 5′-AACGACCCCTTCATTGACCTC-3′, reverse primer: 5′-CCTTGACTGTGCCGTTGAACT-3′.
9
Lrig1 mRNA Expression Quantification
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The total RNA was isolated from the cells using TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. RT-PCR was performed using an SYBR Master Mix kit (Takara, Kusatsu, Japan) on the LightCycler 480 Real-Time PCR System (Roche, Basel, Switzerland). The sequences of primers for the Lrig1 were as follows: forward: 5′-AGCTAACCATCTTATGAGTGCC-3′ and reverse: 5′-CTCAGAAGCAGCAAATTCACA-3′. Each sample was measured in triplicate. A mean value was used to determine mRNA levels using the comparative Ct method, using the formula
, and the mRNA levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH; forward: 5′-GACAAAATGGTGAAGGTCGGT-3′ and reverse: 5′-GAGGTCAATGAAGGGGTCG-3′).
, and the mRNA levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH; forward: 5′-GACAAAATGGTGAAGGTCGGT-3′ and reverse: 5′-GAGGTCAATGAAGGGGTCG-3′).
10
MRPL12 mRNA Expression Analysis
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Total RNAs were extracted from cells with the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and then reverse-transcribed into cDNA using an M-MLV Reverse Transcription Kit (Promega, Madison, WI, USA). A PCR assay was performed using an SYBR Master Mix Kit (Takara, Shiga, Japan) on a LightCycler 480 II real-time PCR instrument (Roche, Basel, Switzerland). Primer sequences: MRPL12 forward, 5′-CTTGTGCCGATGGGTGGT-3′, reverse, 5′-TCAGGCGGACGGTGAAAT-3′. MRPL12 mRNA expression was calculated via the 2−ΔΔCT method.
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