The total RNA of the samples was extracted using the TRIzol reagent (Takara Bio Inc., Dalian, China). Reverse transcription of the total RNA was then performed using a PrimeScriptTM cDNA kit (Takara Bio Inc., Dalian, China) according to manufacturer's directions. Real-time quantitative PCR (RT-qPCR) reactions were incubated initially at 95°C for 30 sec, 95°C for 5 sec, and 60°C for 30 sec of 35 cycles using a SYBR Green PCR Master Mix (Takara Bio Inc., Dalian, China) and were carried out with a PCR System 9700 (Applied Biosystems, USA). Three samples of each group were randomly selected for the RT-qPCR experiment. The samples were analyzed in triplicates, and the relative expression of mRNA was determined using glyceraldehyde-3-phosphatedehydrogenase (GAPDH) as an internal control. The relative gene expression was analyzed using the 2−ΔΔCt method. The specific primers are listed in Supplementary Table 2 and the curves of amplification and dissolution of all RT-qPCR experiments are shown in Supplementary Figure 1 .
Primescript cdna kit
Manufactured by Takara Bio
Sourced in China
The PrimeScript™ cDNA Synthesis Kit is a reagent kit used for the reverse transcription of RNA into complementary DNA (cDNA). The kit contains the necessary components, including PrimeScript reverse transcriptase, primers, and reaction buffers, to efficiently synthesize first-strand cDNA from various RNA templates.
Automatically generated - may contain errors
2 protocols using primescript cdna kit
1
Quantitative Analysis of Gene Expression
2
Quantitative Real-Time PCR Analysis
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Total RNA was extracted from cell samples using Trizol reagents (Thermo Fisher Scientific, Waltham, MA, USA) from cell lines, and cDNA was synthesized using PrimeScriptTM cDNA Kit (Takara, Dalian, China) according to the manufacture’s protocols. qRT-PCR was determined using an ABI 7000 Prism Step One plus detection system (Life Technologies, USA). The relative expression was normalized using GAPDH as an internal reference gene, and U6 was used as the endogenous control of miR-140-5p. Fold changes were calculated using the formula 2-ΔΔCt. All qRT-PCR reactions were performed three times independently. The primer sequences used for qRT-PCR as follow.
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