The expression levels of Collagen I (Col I), Collagen III (Col III), α-smooth muscle actin (α-SMA), Scleraxis (Scx), Tenascin C (TnC) and TGF-β1 were determined by qPCR. Briefly, Cell sample RNAs were extracted using TRIzol reagent (Tiangen Biotech, Beijing, China). Then RNA was converted to cDNA using a Prime-Script™ One Step RT-qPCR kit (Takara Biotechnology Co., Ltd., Dalian, China). PCR reaction was performed using SYBR Green Master Mix (Solarbio Science & Technology Co., Ltd., Beijing, China) in an Applied Biosystems 7500 Real Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). GAPDH was used as the internal control. The relative expression level was calculated by the 2−ΔΔCt method.
Prime script one step rt qpcr kit
Manufactured by Takara Bio
Sourced in China
The Prime-Script™ One Step RT-qPCR kit is a reagent system for reverse transcription and real-time quantitative PCR (RT-qPCR) in a single reaction. It enables the conversion of RNA to cDNA and subsequent amplification and detection of target sequences.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Applied biosystems 7500 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Tiangen Biotech (2 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Sybr green master mix, by Solarbio (1 mentions)
Abi prism 7300 sequence detection system, by Thermo Fisher Scientific (1 mentions)
Rnaiso plus kit, by Takara Bio (1 mentions)
Sybr premix ex taq, by Takara Bio (1 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (1 mentions)
Lightcycler 480, by Roche (1 mentions)
Dna master sybr green 1 kit, by Roche (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Thermal cycler dice real time system 2, by Takara Bio (1 mentions)
Rox reference dye 2, by GenStar (1 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Sybr premix ex tagtm, by Takara Bio (1 mentions)
G8772, by Merck Group (1 mentions)
Cybio felix, by Analytik Jena (1 mentions)
7 protocols using prime script one step rt qpcr kit
1
Quantification of Extracellular Matrix Markers
2
Quantifying Caspase-1 mRNA Expression in Serum
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All 136 patients in the two groups were measured for their mRNA expression of caspase-1 using RT-qPCR. For total RNA extraction from serum samples, the present study used the RNAiso Plus kit (Takara Bio, Inc.), and subsequently transcribed it into cDNA using the Prime-Script™ one-step RT-qPCR kit (Takara Bio, Inc.) according to the manufacturer's protocols. The RT-qPCR process was performed on the ABI PRISM7300 Sequence Detection System (Applied Biosystems; Thermo Fisher Scientific, Inc.) using the SYBR Premix ExTaq (Takara Bio, Inc.). The thermocycling conditions used for qPCR were as follows: An initial activation step at 95˚C for 15 min, followed by 35 cycles of denaturation at 94˚C for 15 sec, annealing at 55˚C for 25 sec and extension at 70˚C for 30 sec. Primer sequences for caspase-1 were as follows: Forward, 5'-CACACCGCCCAGAGCACAAG-3', and reverse, 5'-TCCCACAAATGCCTTCCCGAATAC-3'. Primer sequences for GAPDH were as follows: Forward, 5'-ACACCATGTATTCCGGGTCAAT-3', and reverse, 5'-CCACCACCCTGTTGCTGTAG-3'. GAPDH was utilized as an internal control. Expression levels of caspase-1 were calculated using the 2-ΔΔCq method (25 (link)).
3
Quantification of XIST, miR-133a, and SOCS2
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The expression levels of XIST, miR-133a, and SOCS2 were measured using qRT-PCR. In brief, the extraction of total RNA was conducted using the TRIzol reagent (Tiangen Biotech, Beijing, China), and the mirVana miRNA isolation kit (Ambion, Austin, TX, USA) was used to extract the miRNA. A PrimeScript One Step RT-qPCR kit (Takara Biotechnology, Dalian, China) was used to convert RNA to cDNA for mRNA, and a TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems Life Technologies) was used to convert RNA to cDNA for miRNA, respectively. The PCR analysis was performed in an Applied Biosystems 7500 Real Time PCR system (Applied Biosystems, Thermo Fisher Scientific). All primers used in PCR were designed and synthesized by GeneChem. GAPDH and U6 small nuclear RNA (U6 snRNA) were used as internal references for mRNA and miRNA, respectively. The relative expression level was calculated by the 2−ΔΔCq method.27 (link)
4
Gene Expression Analysis of Metabolic and Inflammatory Markers
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Total RNA was extracted from cells using TRIzol reagent (Invitrogen). After total RNA was reverse transcribed into cDNA by using a Prime-Script™ One Step RT-qPCR kit (Takara Biotechnology Co., Ltd., Dalian, China), PCR reactions for SIRT1, PGC-1α, PPAR-γ, IL-1β, IL-6 and TNF-α mRNA were performed on a LightCycler 480 (Roche, Mannheim, Germany) system with GAPDH used as an internal control. The light cycler DNA master SYBR green I kit (Roche Molecular Biochemicals, Mannheim, Germany) was then used for PCR experiments. The following primers were used: SIRT1 Forward: 5′-GGTGTTAAATACCAAACTGC-3′ and reverse: 5′-AGGAGTGATGTTCAAAATG-3′; PGC-1α Forward: 5’-AATTCACAATCACAGGATCAGAACA3’ and reverse: 5’-ACTTAAGGTGCGTTCAATAGTCTT-3’; PPAR-γ Forward: 5’-TTGGCCATATTTATAGCTGTCATTATT-3′ and reverse: 5’- TGTCCTCGATGGGCTTCA-3’; TNF-α Forward: 5’-GAGCTGTGGGGAGAACAAAAGGA-3′ and reverse: 5’- TTGGCCCTTGAAGAGGACCTG-3’; IL-1β Forward: 5’-GAC CTT CCA GGA TGA GGA CA-3′ and reverse: 5’-AGC TCATATGGGTCCGACAG-3’; IL-6 Forward: 5’-TCC AGT TGC CTTCTT GGG AC-3′ and reverse: 5’-GTGTAATTAAGCCTCCGACTTG-3’; GAPDH Forward: 5’-AGAAGGCTGGGGCTCATTTG-3’ and reverse: 5’-AGGGGCCATCCACAGTCTTC-3. The relative expression of the target genes was calculated using the 2−ΔΔCt method. All experiments were repeated in triplicate.
5
Quantification of miR-2355-3p, circRBMS1, and MST1
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Levels of miR-2355-3p, circRBMS1, and MST1 were detected using qRT‐PCR. Extraction of total RNA from HCMs and tissues were conducted by TRIzol reagent (Invitrogen). For miR-2355-3p quantification, 2 μg of template RNA was reversely transcribed into cDNA using TransScript First-Strand cDNA Sythesis SuperMix. The fluorescence quantitative PCR reaction was performed on Thermal Cycler Dice Real-Time System II (Takara). For quantification of circRBMS1 or MST1, Prime-Script™ One Step RT-qPCR kit (Takara) was applied for reverse transcription from RNA into cDNA. The PCR reactions were performed on an ROX Reference Dye II (GenStar) on StepOnePlusTM Real-Time PCR System (Applied Biosystems). The primer sequences used in qPCR were as follows: U6 forward 5'-CTCGCTTCGGCAGCACA-3' and reverse 5'- AACGCTTCACGAATTTGCGT-3'; GAPDH forward 5'-CACCAGGGCTGCTTTTAACTC-3’ and reverse 5'-TGGAAGATGGTGATGGGATTT-3'; miR‐2355‐3p forward 5'-CTGAGGGATCCCCAGATACAATGG-3' and reverse 5'-GTGCAG GGTCCGAGGT-3'; circ-RBMS1: forward 5'-CCCTGATCTCCATACCCAGA-3' and reverse 5'-TGGAGTCGAGTGTTTGCAGT-3'; MST1 forward 5'-AGACCTCCAGGAGATAATCAAAGA-3' and reverse 5'-AGATACAGAACCAGCCCCACA-3'. The expression of mRNA and miRNA was normalized to GAPDH and U6, respectively. Quantification of RNA was analyzed using 2−ΔΔCt method.
6
Quantitative Analysis of miR-374a-5p and ZEB1
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Total RNA was extracted from serum and cultured cells using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. RT was conducted using a Taqman MicroRNA Reverse Transcription Kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) for miRNA or a Prime-Script™ One Step RT-qPCR kit (Takara Bio, Inc.) for mRNA strictly according to the manufacturers' instructions. Real-time PCR was conducted using SYBR Premix ExTagTM (Takara Bio, Inc.) on a 7500 Fast Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) with the following thermocycling conditions: 95˚C for 10 min, followed by 40 cycles at 94˚C for 45 sec, 55˚C for 30 sec and 72˚C for 1 min. Relative expression levels of miR-374a-5p and ZEB1 were calculated using the 2-ΔΔCq method using U6 snRNA or GAPDH as internal controls (29 (link)). The primers used were as follows: miR-374a-5p forward, 5'-GCGCGCTTATAATACAACCTGA-3' and reverse, 5'-AGAGCAGGGTCCGAGGT-3' (universal); ZEB1 forward, 5'-GATGATGAATGCGAGTCAGATGC-3' and reverse, 5'-ACAGCAGTGTCTTGTTGTTGT-3'; U6 forward, 5'-CAGCACATATACTAAAATTGGAACG-3' and reverse, 5'-ACGAATTTGCGTGTCATCC-3'; GAPDH forward, 5'-TGTGGGCATCAATGGATTTGG-3' and reverse, 5'-ACACCATGTATTCCGGGTCAAT-3'.
7
Quantitative Analysis of Plasmid-Derived RNA
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Cells were grown in a 96-well deep-well plate (Corning, CLS3961) supplied with a sterile 4-mm glass bead in each well to avoid cell clumping. One-milliliter cell cultures were collected by centrifugation (3,000 × g for 3 min at room temperature) and resuspended in 0.3 mL ice-cold Tris-buffer (50 mM Tris-HCl, pH8.0, 100 mM NaCl). 0.5 mm acid-washed glass beads (Sigma, G8772) were added to the cell suspension and then cells were disrupted by RETSCH MM400 Mixer Mill with 5 cycles of mixing at the top speed (60/s) for 30 s. The resultant supernatant was used for 96-well nucleic acid extraction with the combined use of Quick- ‐DNA/RNA Viral MagBead Automated Kit (Zymo Research, R2141) and CyBio Felix automated liquid handler (Analytik Jena). Two microliters of total nucleic samples was subjected to qPCR analysis in a 10-µL reaction volume using PrimeScript OneStep RT-qPCR Kit (TaKaRa, RR064). For the quantification of RNA transcribed from a launching plasmid, RT-qPCR was performed with and without reverse transcriptase. The relative transcript level per plasmid was calculated by using the following formula:
Replicated RNA level was quantified by using 25S rRNA as a reference gene.
Replicated RNA level was quantified by using 25S rRNA as a reference gene.
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