qRT-PCR was performed as described previously36 (link). Total RNA of tissue specimens was isolated using TrizolTM (Life Technology, USA) according to the manufacturer’s instructions. First-strand cDNA was synthesized using MLV transcriptase Kit (Life Technology, USA). qRT-PCR was performed using gene specific primers (Supplementary Table 1 ) for indicated genes with SYBR® Premix Ex Taq™ II (Takara, Japan). The PCR condition was as follows: 95 °C for 4 min, followed by 45 cycles of 95 °C for 20 s, 60 °C for 20 s and 70 °C for 30 s.
Mlv transcriptase kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The MLV transcriptase Kit is a laboratory product designed for the reverse transcription of RNA to cDNA. It contains the Moloney Murine Leukemia Virus (MLV) reverse transcriptase enzyme, necessary buffers, and other essential components required for the reverse transcription process.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (3 mentions)
Sybr premix ex taq 2, by Takara Bio (1 mentions)
Protein a g coupled dynabeads, by Thermo Fisher Scientific (1 mentions)
Platinum sybr green qpcr supermix udg with rox, by Thermo Fisher Scientific (1 mentions)
Bulge loop mirna qrt pcr primer, by RiboBio (1 mentions)
Abi prism 7500 sequence detection system, by Thermo Fisher Scientific (1 mentions)
Cfx96 real time quantitative pcr detection system, by Bio-Rad (1 mentions)
4 protocols using mlv transcriptase kit
1
Quantitative Real-Time PCR Protocol
2
Immunoprecipitation and qRT-PCR Analysis
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Cells were lysed in NP-40 lysis buffer (50 mM Tris-HCl, ph 7.4, 150 mM NaCl, 1% NP-40 and Protease inhibitor cocktail and RNAse inhibitor) and cleared lysates were immunoprecipitated with indicated anti-Bmi1 and IgG antibodies. Immune complexes were purified with Protein-A/G-coupled dynabeads (Life Technologies). Immunoprecipitated and input RNA were isolated by TrizolTM LS reagent. RNA was reverse transcribed by MLV transcriptase Kit (Life Technologies). cDNA was used as a template in qRT–PCR amplifications with PANDAR specific primers.
3
Quantification of miR-148b Expression
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Total RNA was extracted using TRIzol (Invitrogen, USA) according to the manufacturer’s protocol. cDNA was synthesized with the MLV transcriptase Kit (Invitrogen, USA). The quantitative analysis of miR-148b expression was assayed using a Bulge-Loop TM miRNA qRT-PCR primer (forward, 5′-ACACTCCAGCTGGGTCAGTGCATC-3′ and reverse, 5′-CTCAACTGGTGTCGTGGA-3′; RiboBio, China) and Platinum® SYBR® Green qPCR SuperMix-UDG with ROX (Invitrogen, USA) on an ABI PRISM® 7500 Sequence Detection System (Applied Biosystems, Foster, CA). U6 small nuclear RNA (forward, 5′- CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′) was used as an internal control (RiboBio, China). The fold changes were calculated through relative quantification using the 2-ΔΔCt method (18).
4
Real-time qPCR Analysis of CRC Cells
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Total RNA was extracted from HCT116 CRC cells using TRIzol® (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. cDNA was synthesized using the MLV transcriptase kit (Invitrogen; Thermo Fisher Scientific, Inc.). Fast SYBR Green master mix was used to determine the threshold cycle (Cq) value of each sample using a CFX96 real-time quantitative PCR detection system (Bio-Rad Laboratories, Inc.). GAPDH served as the gene used for normalization. The fold-changes were calculated using the relative quantification with 2−ΔΔCq (29 (link)). All reactions were performed in a 20 µl reaction volume in triplicate. The following PCR conditions were used: Initial denaturation at 95°C for 30 sec; followed by 40 cycles of 95°C for 5 sec and 60°C for 30–60 sec; and stage 3 was dissociation according to the manufacturer's protocol (cat. no. RR420; Takara Biotechnology Co., Ltd.).
The PCR primer sequences used were as follows: GAPDH forward, 5′-AAGGTCATCCCTGAGCTGAA-3′ and reverse, 5′-TGACAAAGTGGTCGTTGAGG-3′; G6PD forward, 5′-TGCATGAGCCAGATAGGCTG-3′ and reverse, 5′-GGTAGTGGTCGATGCGGTAG-3′; and PKM2 forward, 5′-ATGCAGCACCTGATAGCTCG-3′ and reverse, 5′-AGGCTCGCACAAGTTCTTCA-3′.
The PCR primer sequences used were as follows: GAPDH forward, 5′-AAGGTCATCCCTGAGCTGAA-3′ and reverse, 5′-TGACAAAGTGGTCGTTGAGG-3′; G6PD forward, 5′-TGCATGAGCCAGATAGGCTG-3′ and reverse, 5′-GGTAGTGGTCGATGCGGTAG-3′; and PKM2 forward, 5′-ATGCAGCACCTGATAGCTCG-3′ and reverse, 5′-AGGCTCGCACAAGTTCTTCA-3′.
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