Total RNA was extracted from the mouse hearts using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), and cDNAs were produced by reverse-transcribing RNA using the Prime Script RT reagent kit (TaKaRa, Otsu, Japan). qRT-PCR was performed using SYBR Green dye (Kapa Biosystems, Wilmington, MA, USA), and gene expression was normalized to β-actin. The sequences of the specific primers for each of the transcripts are shown in Table A in S1 File .
Sybr green dye
Manufactured by Roche
Sourced in United States, Germany, Switzerland, China
SYBR Green dye is a fluorescent dye used in molecular biology for the detection and quantification of DNA. It binds to double-stranded DNA, causing it to emit a green fluorescent signal when exposed to blue or ultraviolet light. The intensity of the fluorescent signal is proportional to the amount of DNA present, making it a useful tool for applications such as real-time PCR, gel electrophoresis, and DNA sequencing.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (16 mentions)
Rneasy mini kit, by Qiagen (7 mentions)
Trizol, by Thermo Fisher Scientific (5 mentions)
Lightcycler 480, by Roche (5 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (4 mentions)
Superscript 3, by Thermo Fisher Scientific (4 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Iq5 multicolor real time pcr detection system, by Bio-Rad (4 mentions)
Primescript rt reagent kit, by Takara Bio (3 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
7500 real time system, by Thermo Fisher Scientific (3 mentions)
7900 sequence detector, by Thermo Fisher Scientific (3 mentions)
Primescript rt reagent kit, by Roche (3 mentions)
Reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Rneasy kit, by Qiagen (3 mentions)
Superscript 2 rt, by Thermo Fisher Scientific (3 mentions)
Reverse transcription kit, by Takara Bio (3 mentions)
Rneasy mini kit, by Thermo Fisher Scientific (3 mentions)
Hs rnu6b 2 miscript primer assay, by Qiagen (2 mentions)
Miscript sybr green pcr kit, by Qiagen (2 mentions)
62 protocols using sybr green dye
1
Mouse Heart RNA Extraction and qRT-PCR Analysis
2
qRT-PCR Analysis of Mouse Heart RNA
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After extraction of total RNA from the mouse hearts using the TRIzol reagent (Invitrogen Life Technologies, USA), RNA was reverse-transcribed to produce cDNAs using the Prime Script RT reagent kit (TaKaRa, Japan). cDNA was used as a template for qRT-PCR using SYBR green dye (Kapa Biosystems, USA), and gene expression was normalized to β-actin. The sequences of the specific primers are described in Supplementary Table S1 .
3
Quantifying Gene Expression in Cells
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Total RNA from the cells was extracted by TRIzol (Invitrogen), and the synthesis of cDNA was performed using an oligo(dT) primer by ReverAid™ Premium First Strand cDNA Synthesis Kit (Fermentas Life Science), according to the manufacturers' protocols. The quantification of gene transcripts was analysed by real-time PCR using SYBR green dye (KAPABIOSYSTEMS). All values were normalized to the expressing level of Actin. The primers were as follows:
For human samples: Actin forward, CCAGAGGAAGAGAGGCATCC; Actin reverse, GTGGTGGTGAAGCTGTAGCC. V-Src forward, TTCGGGGACTTCAACACTTC; V-Src reverse, ATGAGCCAGCCACCAGTTAC; H-Ras forward, TGCCATCAACAACACCAAGT; H-Ras reverse, ATCTCACGCACCAACGTGTA.
For mouse sample, p21 forward, AAGTGTGCCGTTGTCCTTC; p21 reverse, ACTTCAGGGTTTTCTCTTGC Actin forward, AGCCATGTACGTAGCCATCC; Actin reverse, CTCTCAGCTGTGGTGGTGAA.
For human samples: Actin forward, CCAGAGGAAGAGAGGCATCC; Actin reverse, GTGGTGGTGAAGCTGTAGCC. V-Src forward, TTCGGGGACTTCAACACTTC; V-Src reverse, ATGAGCCAGCCACCAGTTAC; H-Ras forward, TGCCATCAACAACACCAAGT; H-Ras reverse, ATCTCACGCACCAACGTGTA.
For mouse sample, p21 forward, AAGTGTGCCGTTGTCCTTC; p21 reverse, ACTTCAGGGTTTTCTCTTGC Actin forward, AGCCATGTACGTAGCCATCC; Actin reverse, CTCTCAGCTGTGGTGGTGAA.
4
Quantification of miR-185 and Hypertrophy Markers
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qRT-PCR for miR-185 targets and hypertrophic markers was performed with primers listed in Table B in S1 File , using SYBR green dye (Kapa Biosystem) and StepOne Plus Real Time PCR System (Applied Biosystems). miRNA-specific qRT-PCR in tissue or isolated cells was done using miScript SYBR Green PCR Kit (Qiagen) according to the manufacturer’s protocol, with miScript Primer Assay (for miR-185; Qiagen). The expression of the mRNAs was normalized to 18S rRNA and the level of miR-185 was normalized to U6 small RNA using the Hs_RNU6B_2 miScript Primer Assay (Qiagen). All reactions were performed in triplicate.
5
Reverse Transcription and qRT-PCR
6
Quantitative Analysis of mRNA and miRNA Expression
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Analysis and quantification of mRNA expression levels were performed with the primers listed in Supplementary Table S3 , using SYBR green dye (Kapa Biosystems, Boston, MA, USA) and StepOne Plus Real Time PCR System (Applied Biosystems, Waltham, MA, USA). miRNA-specific qRT-PCR in tissue or isolated cells was performed using a miScript SYBR Green PCR kit (Qiagen) according to the manufacturer’s instructions, with miScript Primer Assay (Qiagen). The expression of mRNAs was normalized to 18S rRNA and the level of mature miRNA was normalized to U6 small RNA using the Hs_RNU6B_2 miScript Primer Assay (Qiagen), respectively. All reactions were performed in triplicate.
7
Real-Time PCR Analysis of Apoptosis-Related Genes
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Real-time PCR was performed by use of the SYBR Green dye (KapaBiosystems, Inc. of Wilmington, MA, USA) on the Exicycler™96 Bioneer machine (Bioneer Corporation, Daejoen, Korea). Each PCR reaction took place in triplicate. The forward and reverse primers used for PCR amplification of Bax, Bcl-2, p53, Cathepsin B, Caspase-3, Caspase-9 and EF-2 gene are all listed in Table 1. The PCR reactions were made by mixing 2X KAPA SYBR® FAST qPCR Master Mix (KapaBiosystems, Inc. of Wilmington, MA, USA) with each primer and cDNA template. The real-time PCR program was carried out with a following 1 cycle of initial denaturation at 94°C for 10 min through 40 cycles of PCR reaction and then held for 30 seconds at each temperature level. Finally, melting curve analysis was performed over a gradient extending from an annealing to a denaturation temperature. The expression was calculated by using the relative standard curve method of quantification and reported as a fold change of gene expression.
8
Quantitative RT-PCR Expression Profiling
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1 µg of total RNA was reverse transcribed to cDNA with random primers using high capacity cDNA reverse transcription kit (New England, USA) as per manufacturer's protocol in a 20 µL volume. cDNA was diluted 1:10 with nuclease free water to a final concentration of 5 ng/µL and stored at -20 °C until the next use. All RT-qPCR reactions were performed on ABI7900 HT Fast Real Time PCR Instrument (Thermo Fisher Scientific) in duplicate, except the samples in which the analysis outcome was questionable. If this had happened, another two replicates were analysed. Detection of the amplification product was enabled with SYBR Green dye (Roche-OR, USA), according to the manufacturer's recommendations, in total reaction volume of 10 μl. Expression levels were normalized using 18S gene (housekeeping gene). Relative expression was calculated with the mathematical model allowing for correction of reaction efficiency and using the Universal Human Reference RNA (Stratagene, La Jolla, CA, USA) as a reference. Primer sequences used in this study are shown in Table 1; detailed PCR protocols are available upon request from the corresponding author.
9
Quantitative PCR Analysis of Gene Expression
10
Quantification of miR-519 and PD-L1 Expression
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Total RNA was extracted as previously described. Takara- PrimeScriptTM RT reagent Kit (Takara Bio, Inc.) was used to reverse transcribe RNA into cDNA according to the manufacturer's protocol. The reverse transcription thermocycling program was 37°C for 15 min followed by 85°C for 5 sec. SYBR®-Green dye (Roche Diagnostics) was used to perform qPCR according to the manufacturer's protocol. The thermocycling program was: Step 1, 95°C for 30 sec; step 2, 95°C for 3 sec; step 3, 60°C for 30 sec (step 2–3, 40 cycles); and step 4, holding at 10°C. GAPDH and U6 were utilized as housekeeping genes for the detection of PD-L1 and miR-519, respectively. Relative expression levels of genes were calculated using the 2−ΔΔCt method (20 (link)). The primer sequences were as follows: miR-519 forward, 5′-CATGCTGTGACCCTCCAAAG-3′ and reverse, 5′-GAGAAAACAAACAGAAAGCGCT-3′; PD-L1 forward, 5′-CTGAACGCCCCATACAACAA-3′ and reverse, 5′-CTTGGAATTGGTGGTGGTGG-3′; GAPDH forward, 5′-GAGAAGTATGACAACAGCCTC-3′ and reverse, 5′-ATGGACTGTGGTCATGAGTC-3′; and U6 forward, 5′-CTCGCTTCGGCAGCACATATACTA-3′ and reverse, 5′-ACGAATTTGCGTGTCATCCTTGCG-3′.
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