Four circRNAs were selected from circRNA differential expression profiles for population Q-PCR experiments. Both the Prime Script™ RT reagent Kit with gDNA Eraser and Q-PCR kits were equipped with Takara Bio Inc, Japan. Reverse transcription reaction system: 5×gDNA Eraser Buffer 2.0ul, gDNA Eraser 1.0 ul, add appropriate amount of total RNA and make up to 10ul with enzyme-free water, place at 42 °C and heat for 2 minutes to remove the DNA from the genome, then add to the above liquid PrimeScript RT Enzyme Mix I 1.0 ul, RT Primer Mix 1.0 ul, 5× PrimeScript Buffer 2 (for Real Time) 4.0 ul, RNase Free dH2O 4.0 ul, shake gently and immediately put at 37 °C for 15 minutes. The extension reaction was carried out under the conditions of reverse transcription inactivation in an environment of 85 °C and 5 seconds, and finally it was cooled under the heat preservation condition of 4. Q-PCR reaction system: TB Green Premix Ex Taq (Tli RNaseH Plius) (2X) 10 ul, PCR Forward Primer (10M) 0.8 ul, PCR Reverse Primer (10 M) 0.8 ul, ROX Reference Dye (20X) 0.4 ul, enzyme-free water 6 ul, cDNA 2 ul. Reaction conditions: 95 °C, 34 s cycle to activate enzyme activity once, 95 °C, 5 s, 60 °C, 34 s for 40 cycles of extension reaction, using housekeeping gene GAPDH as internal reference, and finally set the melting curve analysis. Table 1 is the primer sequence of target circRNA and GAPDH.
Qpcr kits
Manufactured by Takara Bio
Sourced in Japan
The QPCR kits from Takara Bio are designed for the quantitative polymerase chain reaction (qPCR) technique. These kits provide the necessary reagents and components to perform real-time PCR analysis for the quantification of specific DNA or RNA targets.
Automatically generated - may contain errors
Lab products found in correlation
Primescript rt reagent kit with gdna eraser, by Takara Bio (1 mentions)
Superscript 4, by Thermo Fisher Scientific (1 mentions)
Abi 7900 fast real time detection system, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Gotaq qpcr master mix, by Promega (1 mentions)
Tnf α quantikine elisa kit, by R&D Systems (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Sybr premix ex taq 2, by Takara Bio (1 mentions)
5 protocols using qpcr kits
1
Profiling of Circulating circRNAs by Q-PCR
2
Quantitative Analysis of CAR Gene Expression
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Total DNA extraction and qPCR were performed using MiniBEST Whole Blood Genomic DNA (9781, Takara) and qPCR kits (RR390, Takara). CAR F-primer: 5′-CACCGATTTTACCCTGAC -3′, and R-primer: 5′-GCCAAAGGTAGGAGGAATA -3′; MGB-probe：5′−6-VIC-AAGACTTCGCCGTGTATTA -MGB-3′.
3
qPCR Gene Expression Analysis
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The RNA extraction process was performed, and subsequently, cDNA was synthesized following the guidelines (Toyobo, Japan), employing a reverse transcription kit (SuperScript IV, Thermo Fisher Scientific). qPCR kits (Takara, Japan) were employed to measure the expression levels of mRNAs. The experiment involved a reaction volume of 20 μL. The reaction process consisted of an initial step of heating at 95°C for 10 min, followed by 40 cycles of a 5 s heating period at 95°C. At 60°C for 30 s, the primers’ sense and antisense details can be found in the supplemental materials (Table S2 ), and G3PDH was used as a control. The relative expression level was determined using the 2−ΔΔCt method after incubating for 30 s at 72°C.
4
Quantifying Gene Expression in Lung Tissue
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Total RNA was extracted from pulmonary tissues using TRIzol reagent (Invitrogen Life Technologies) according to the manufacturer’s instructions. qPCR kits (Takara, Kyoto, Japan) were used for the qPCR experiment. Total RNA (4 μg) templates were used to make cDNA by using AMV reverse transcriptase and random primers (9-mer) as the first strand primer. Synthesized cDNA was used in qPCR experiments. qPCR was performed using a 40-cycle two-step PCR with sequence-specific primer pairs using the ABI 7900 fast real-time detection system (Invitrogen Life Technologies). The qPCR cycling conditions were: 95°C for 10 min, 40 cycles of 95°C for 15 sec and 60°C for 1 min, then 95°C for 1 min, followed by dissociation curve analysis. The qPCR reagents used in were from the GoTaq® qPCR Master Mix (A6001; Promega Corporta). Primers were designed using the Primer Express 3.0 software and the sequences were as follows: MMP9: Forward, 5′-AAAGACCTGAAAACCTCCAACCT-3′, and reverse, 5′-GCCCGGGTGTAACCATAGC-3′; TNF-α: forward, 5′-GAAGTTCCCAAATGGCCTCC-3′, and reverse, 5′-GTGAGGGTCTGGGCCATAGA-3′; P65: forward, 5′-GGTCCACGGCGGACCGGT-3′, and reverse, 5′-GACCCCGAGAACGTGGTGCGC-3′. The levels of mRNA expression were evaluated as a ratio based on the qPCR results for lung tissue GAPDH mRNA using the 2−ΔΔCt method.
5
Quantifying NE, TNF-α, and Protein Expression
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NE and TNF-α concentration in heart tissues, perfusate, and cell supernatants were determined using the NE research enzyme-linked immunosorbent assay (ELISA) kit (ALPCO#17-NORHU-E01-RES) and the TNF-α Quantikine ELISA kit (R&D System#RTA00), respectively. The western blotting assays of proteins were performed using standard protocol22 (link). Antibodies for western blotting assays are available in the Supplementary Methods. Quantitative RT-PCR assay of relative mRNA expression was analyzed using standard real-time PCR protocol according to TAKARA qPCR kits. In brief, total RNA was reverse transcribed using a PrimeScriptTM RT Reagent Kit (TAKARA#RR047A). Real-time PCR were performed with the SYBR Premix Ex Taq II (TAKARA#RR820A) in a LightCycler480 real-time PCR system. Primer sequences for genes are shown in Table S3 .
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