Genomic DNA was extracted using Genomic DNA Mini Preparation Kit (Beyotime, Beijing, China) according the instruction. Total RNA was collected using QIAshredder and RNeasy Kit (QIAGEN) according to the manufacturer's instruction. 1.0 ug RNA was reverse‐transcribed using the SuperScript™ III Reverse Transcriptase kit (Life Technologies). PCR was performed on the first strand of cDNA with the primers listed in Table 2 using the Phusion reaction system (Thermo Scientific, Waltham, MA) or rTaq (TaKaRa).
Phusion reaction system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Phusion reaction system is a laboratory equipment designed for performing high-fidelity PCR amplifications. It features a high-performance thermostable DNA polymerase that ensures accurate DNA synthesis with minimal error rates.
Automatically generated - may contain errors
Lab products found in correlation
Qiashredder and rneasy kit, by Qiagen (2 mentions)
Superscript 3 reverse transcriptase kit, by Thermo Fisher Scientific (2 mentions)
Genomic dna mini preparation kit, by Beyotime (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Qiashredder, by Qiagen (1 mentions)
One step sybr primescript rt pcr kit 2, by Takara Bio (1 mentions)
Superscript 3 reverse transcriptase, by Takara Bio (1 mentions)
Hairpin it mirna rt pcr quantitation kit, by GenePharma (1 mentions)
Rt pcr system, by Bio-Rad (1 mentions)
Sybr premix ex taq 2, by Takara Bio (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
4 protocols using phusion reaction system
1
Genomic DNA and RNA Extraction for Reverse Transcription and PCR
2
Total RNA Extraction and RT-PCR Analysis
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Total RNA was extracted by using QIAshredder and RNeasy Kit (QIAGEN) according to the manufacturer's protocol. 1.0 µg RNA was reverse-transcribed with the SuperScript™ III Reverse Transcriptase kit (Thermo Scientific) following the manufacturer's protocol. PCR was performed by using the Phusion reaction system (Thermo Scientific) and RT-PCR was performed by using rTaq system (Thermo Scientific) with the primers listed in Table 2 .
3
Quantification of miRNA and mRNA Expression
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Total RNA from cells, tumor tissues and paired adjacent non-tumor tissues were extracted using the QIAshredder and RNeasy kit (Qiagen GmbH). Accordingly, 1 mg RNA was reverse-transcribed to cDNA using SuperScript™ III Reverse Transcriptase (Takara Bio, Inc.). PCR was performed using the Phusion reaction system according to the manufacturer's protocol (Thermo Fisher Scientific, Inc.). miR-383 cDNA was synthesized using Hairpin-it™ miRNA RT-PCR Quantitation kit (Shanghai GenePharma, Co., Ltd.) at 25°C for 30 min, 42°C for 30 min and 85°C for 5 min. qPCR was performed on a RT-PCR system (Bio-Rad Laboratories, Inc.). The thermocycling conditions were: 95°C for 3 min; followed by 40 cycles of at 95°C for 12 sec and 60°C for 40 sec. The expression levels of miR-383 were normalized to U6. Detection of RBM24 mRNA was performed using a One Step SYBR PrimeScript™ RT-PCR kit II (Takara Bio, Inc.) and GAPDH expression was used as the loading control. The relative quantification of gene expression was determined using the comparative CT method (2−∆∆Cq) (24 (link)). Primers used for RT-qPCR are listed in Table II .
4
Cardiomyocyte RNA Extraction and qRT-PCR Analysis
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Total RNA was extracted from cardiomyocytes using TRIzol reagent (Invitrogen, USA). The concentration and quality of RNA were determined. The RNA integrity was determined using agarose gel electrophoresis. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an endogenous control. According to the reverse transcription kit instructions, 500 ng of RNA template was added, and cDNA was obtained by RT using a 20 µL reaction system. The RT conditions were 37 ℃ for 15 minutes, 85 ℃ for 5 seconds. Quantitative (q)RT-PCR was performed using a Phusion reaction system (Thermo Fisher Scientific, Waltham, MA, USA) and SYBR PreMix Ex Taq™II (Takara, Shiga, Japan) for qRT-PCR. reaction conditions were 95 ℃ for 30 seconds, 40 cycles of 95 ℃ for 5 seconds, and 60 ℃ for 30 seconds. The primer sequences for each gene are shown in Table 1 .
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