Total RNAs of tissues or cell lines were extracted using TRIzol reagent according to manufacturer’s protocol. The cDNA was transcribed using a Prime Script™ RT Master Mixture according to the manufacturer’s protocol (Takara Biotechnology, Daliang, China). qPCR was performed on an ABI Prism PCR system and the expression of miR-21-5p was quantified via the SYBR Green I fluorescent dye method (Takara Biotechnology). PCR was initiated at 95°C for 2 min, followed by 36 cycles at 95°C for 20 s and 58°C for 25 s. The following forward and reverse primers were used: 5′-GGGGTAGCTTAT CAGACTGATG-3′, 5′-TGTCGTGGAGCGGCAATTG-3′ (miR-21-5p); 5′-CGCTTCGGCACATATACTA-3′, 5′-CGCTTCACGAATTTGCGTGTCA-3′ (U6). The relative level of miR-21-5p was calculated with the comparative 2−ΔΔCt method normalized by U6.
Prime script rt master mixture
Manufactured by Takara Bio
Sourced in China, Japan
Prime Script™ RT Master Mixture is a ready-to-use solution for reverse transcription (RT) of RNA into cDNA. It contains all the necessary components, including a reverse transcriptase enzyme, for efficient and reliable cDNA synthesis.
Automatically generated - may contain errors
Lab products found in correlation
Sybr primescript mirna rt pcr kit, by Takara Bio (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Abi prism pcr system, by Takara Bio (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Takara Bio (1 mentions)
Taqman microrna rt kit, by Thermo Fisher Scientific (1 mentions)
Qrt pcr kit, by Merck Group (1 mentions)
Sybr prime script kit, by Takara Bio (1 mentions)
6 protocols using prime script rt master mixture
1
Quantification of miR-21-5p Expression
2
Quantitative Real-Time PCR for miRNA and mRNA Expression
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Total RNA was extracted in SCC9 and SCC15 cells by the application of TRIzol® reagent (Takara, Japan) in line with the protocols of supplier. Prime Script™ RT Master Mixture (Takara 11141ES10, Japan) or TaqMan® MicroRNA RT kit (4366596, Applied Biosystems™, Foster City, CA, USA) was utilized for RNA reverse transcription (RT). Then, qPCR was implemented with the qRT-PCR Kit (QR0100-1KT, Sigma-Aldrich, USA) by using StepOnePlus™ Real-time PCR Systems (Applied Biosystems™). Relative expression levels were calculated using the 2−ΔΔCt method, with U6 small nuclear RNA (U6) as the endogenous control to normalize miRNA expression and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the endogenous control to normalize mRNA/circRNA expression. Related primer sequences were exhibited in Additional file 8 : Table S1.
3
Quantification of miR-21-5p in TSCC
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Total RNA was extracted from the tissues or cell lines using TRIzol® reagent according to manufacturer's protocol. The cDNA was transcribed using a Prime Script™ RT Master Mixture according to the manufacturer's protocol (Takara Biotechnology Co., Ltd.). miR-21-5p in TSCC was detected using SYBR Prime Script miRNA RT-PCR kit (Takara Biotechnology Co., Ltd.). The thermocycling conditions were as follows: Pre-denaturation at 95°C for 1 min, followed by denaturation at 95°C for 15 sec, annealing at 60°C for 40 sec and extension at 72°C for 15 sec, for a total of 40 cycles. The primer sequences in the present study were as follows: hsa-miR-21-5p forward, 5′-GGGGTAGCTTATCAGACTGATG-3′; hsa-miR-21-5p reverse, 5′-TGTCGTGGAGCGGCAATTG-3′; U6: Forward, 5′-CGCTTCGGCACATATACTA-3′; U6 reverse, 5′-CGCTTCACGAATTTGCGTGTCA-3′; PDCD4 forward, 5′-TGTGCCAACCAGTCCA-3′; PDCD4 reverse, 5′-GATCCTAACTATGATGA-3′; GAPDH forward, 5′-TGTTGCCATCAATGACCCCTT-3′; GAPDH reverse, 5′-CTCCACGACGTACTCAGCG-3′. The expression levels of miRNA-21-5p were calculated using the 2−ΔΔCq method (10 (link)).
4
Quantification of miRNA-145-5p Expression
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Total RNA was extracted from the tissues or cells using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to manufacturer's protocol. Total RNA was reverse transcribed into cDNA using the Prime Script™ RT Master Mixture (Takara Biotechnology Co., Ltd.) according to the manufacturer's protocol. The following thermocycling conditions were used for cDNA: 37˚C for 15 min and 85˚C for 15 sec. Subsequently, qPCR was performed using the SYBR Prime Script miRNA RT-PCR kit (Takara Biotechnology Co., Ltd.). The following thermocycling conditions were used for qPCR: Pre-denaturation at 95˚C for 1 min, followed by 40 cycles of denaturation at 95˚C for 15 sec, annealing at 60˚C for 40 sec and extension at 72˚C for 15 sec. The expression level of miR-145-5p was calculated using the 2-ΔΔCq (15 (link)) method and normalized to the internal reference gene U6. The primers used were as follows: miR-145-5p forward, 5'-ACAC TCCAGCTGGGGTCCAGTTTTCCCAGGA-3' and reverse, 5'-ACACTCCAGCTGGGGTCCAGTTTTCCCAGGA-3'; U6 forward, 5'-CGCTTCGGCACATATACTA-3' and reverse, 5'-CGCTTCACGAATTTGCGTGTCA-3'.
5
Quantifying miR-145-5p Expression
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Total RNA was extracted using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and cDNA was synthesized using Prime Script™ RT Master Mixture (Takara Biotechnology Co., Ltd.). A SYBR Prime Script miRNA RT-PCR kit (Takara Biotechnology Co., Ltd.) was used for the detection and quantitation of miR-145-5p expression. The primers were synthesized by Tiangen Biotech (Beijing) Co., Ltd, and the sequences were as follows: miR-145-5p forward, 5’-CGGTCCAGTTTTCCCAGGA-3’, and reverse, 5’-AGTGCAGGGTCCGAGGTATT-3’. miR-145-5p-loop: 5’-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAGGGAT-3’.
6
RNA Extraction and qRT-PCR Analysis
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The authors extracted the total RNA from cells using TRIzol ® reagent (Invitrogen) according to the manufacturer's instructions. cDNA was synthesized using Prime Script™ RT Master Mixture (Takara). qRT-PCR was performed on ABI 7500 Realtime PCR System (ABI) using SYBR Prime Script kit (Takara). The expression levels of mRNA for the tested genes related to GAPDH were analysed using 2 -ΔΔCt method.
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