Total RNA was isolated from the cells using RNAiso Plus (Takara, Japan) and then reversely transcribed using PrimeScript 1st RT Master Mix (Takara) according to the instructions of the manufacturer. The primer sequences were as follows: β-actin (internal group) forward primer: 5′-CACGATGGAGGGGCCGGACTCATC-3′; reverse primer: 5′-TAAAGACCTCTATGCCAACACAGT-3′ and PFKFB3 forward primer: 5′-ATTGCGGTTTTCGATGCCAC-3′; reverse primer: 5′-GCCACAACTGTAGGGTCGT-3′. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed on StepOne Real-Time PCR System with SYBR Premix Ex Taq (Takara) as described by the manufacturer's instructions. The qRT-PCR reaction consisted of a 95°C denaturation step for 30 s, 40 cycles (95°C for 5 s, 65°C for 30 s, and 60°C for 45 s) and an extension at 72°C for 60 s. The expression of target genes was established using the comparative cycle threshold (Ct; 2−ΔΔCT) method.
Primescript 1st rt master mix
Manufactured by Takara Bio
Sourced in Japan
PrimeScript™ 1st RT Master Mix is a ready-to-use solution for reverse transcription. It contains PrimeScript™ Reverse Transcriptase and necessary components for cDNA synthesis from RNA templates.
Automatically generated - may contain errors
Lab products found in correlation
Rnaiso plus, by Takara Bio (2 mentions)
Stepone real time pcr system, by Takara Bio (2 mentions)
Sybr premix ex taq, by Takara Bio (2 mentions)
Cfx connect real time system, by Bio-Rad (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Sybr primescript rt pcr kit, by Takara Bio (1 mentions)
3 protocols using primescript 1st rt master mix
1
Quantitative RT-PCR of PFKFB3 Expression
2
Quantification of Piezo1 Expression
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Total RNA from human NP cells in different groups was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). qRT-PCR was performed according to the manufacturer's instructions. PrimeScript™ 1st RT Master Mix (TaKaRa Biotechnology, Otsu, Japan) was used to synthesize cDNA from total RNA. qRT-PCR was performed using SYBR PrimeScript RT-PCR Kit (TaKaRa Biotechnology, Otsu, Japan) on the CFX connect™ Real-time system (Bio-Rad, USA). The primers of Piezo1 used for qRT-PCR were forward, 5′-ACTTTCTGGTGACCCTGCAC-3′, reverse, 5′-GGCAGGTACAGCCACTTGAT-3′. The relative RNA expression levels were normalized to GAPDH (forward, 5′-TCAAGAAGGTGGTGAAGCAGG-3′, reverse, 5′-TCAAAGGTGGAGGAGTGGGT-3′). The test was performed in triple replication. The relative Piezo1 expression level was analyzed using the 2−ΔΔCt method.
3
Quantitative RT-PCR Analysis of Notch Pathway Genes
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Total RNA was extracted from cultured cells using RNAiso Plus (Takara Bio). This was followed by cDNA synthesis using PrimeScript™ 1st RT Master Mix (Takara, Japan). The primer sequence was as follows: GAPDH (an internal control), forward primer: 5′-ACCACAGTCCATGCCATCAC-3′, reverse primer: 5′-TCCACCACCCTGTTGCTGTA-3′. NOTCH4, forward primer: 5′-CACTAGGCGAGGACAGCATTG-3′, reverse primer: 5′-ATTCC TGGGGAGGAGTTAGCA-3′. NOTCH1, forward primer: 5′-GAGA AGGGAAGTTGAACGAGCAT-3′,reverse primer: 5′-TGCCTCCG TTTGCCTCTGG-3′. EFNB2, forward primer: 5′-GGTGGTCCTC TTGCTGAAGT-3′, reverse primer: 5′-CGCTGACCTTCTCGTAG TGA-3′. HES1, forward primer: 5′-CTACCCCAGCCAGTGTCAA CA-3′, reverse primer: 5′-AACGCAGTACCGGCGAGTG-3′. HEY1, forward primer: 5′-CGGCAGGAGGGAAAGGCTA-3′, reverse primer: 5′-CGGGTGATGTCCAAAGGCAG-3′. Quantitative RT-PCR (qRT-PCR) was performed on the StepOne™ Real-Time PCR System with SYBR Premix Ex Taq™ (TaKaRa, Japan) according to the manufacturer's protocol. The qRT-PCR reaction consisted of a 95°C denaturation step for 30 s, 40 cycles (95°C for 5 s, 65.5°C for 30 s, 60°C for 45 s) and extension at 72°C for 60 s. The expression of target genes was calculated using the 2 -ΔΔCT method.
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