Total RNA was extracted with TRIzol reagent (Invitrogen) and used for first-strand cDNA synthesis using PrimeScript II RT reagent kit (Takara, Madison, WI). SYBR-green Premix Ex-Tag II (Takara) was used for the quantification of SOX4 and EMT markers with a real time quantitative PCR with Prism 7900HT sequence detection system (Applied Biosystems, Foster City, CA). The primer sequences for this study were listed in Table S1. The data was normalized with cyclophilin as control. Quantification of the mature form of miRNAs were performed using the Taqman MicroRNA Assay kit (Applied Biosystems) according to the manufacturer's protocol and RNU6B was used as the internal control. TaqMan probe sequences for this study were listed in Table S2. Data were analyzed using the comparative Ct method. The experiments were performed in triplicates and expressed as the mean ± S.D.
Primescript 2 rt reagent kit
Manufactured by Takara Bio
Sourced in Japan
The PrimeScript II RT Reagent Kit is a reverse transcription kit used for the conversion of RNA to cDNA. The kit includes the necessary reagents for the reverse transcription reaction, including the PrimeScript II Reverse Transcriptase enzyme.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Taqman microrna assay kit, by Thermo Fisher Scientific (1 mentions)
Prism 7900ht sequence detection system, by Thermo Fisher Scientific (1 mentions)
Sybr green premix ex tag 2, by Takara Bio (1 mentions)
Trizol solution, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Prism 7900ht, by Thermo Fisher Scientific (1 mentions)
Sybr premix ex taq, by Takara Bio (1 mentions)
3 protocols using primescript 2 rt reagent kit
1
Quantifying EMT Markers via qPCR
2
Quantifying Gene Expression by RT-qPCR
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Total RNA was prepared using TRIzol solution (Invitrogen), as described by the manufacturer’s instructions. RNA was converted to cDNA by reverse transcription using a PrimeScript II RT Reagent Kit (Takara, Kyoto, Japan). SYBR-Green Premix Ex-Tag™ (Takara) was used for quantifying gene expression using real-time quantitative PCR using the Prism 7900HT sequence detection system (Thermo Scientific) according to manufacturers’ protocols. Oligonucleotides used for the analysis of gene expression are listed in Supplementary Table S2 . The expression of each gene was quantitated using the 2−∆∆CT method and normalized using cyclophilin. Data were analyzed in at least triplicates and expressed as mean ± standard deviation (SD).
3
Quantification of Gene Expression via qRT-PCR
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Total RNA was isolated using Trizol (Invitrogen, Carlsbad, CA, USA) reagent according to the manufacturer’s instructions. Then, RNA was transformed to cDNA with reverse transcription using a PrimeScript II RT Reagent Kit (Takara, Kyoto, Japan). Quantification of specific gene expression was measured with SYBR premix Ex Taq™ (Takara) and a Prism 7900HT sequence detection system (Thermo Fisher Scientific, Rockford, IL, USA) following the manufacturer’s protocols. Relative gene expression was determined using the 2ΔCt (internal control) − ΔCt (gene) method and normalized to cyclophilin [53 (link)]. Data was acquired from at least triplicate experiments and denoted as mean ± standard deviation (SD). Used primer sequences are listed in Supplementary Table S2 .
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