Total RNA was isolated from cells and the testes of rats by TRIZOL reagent (TaKaRa, Dalian, China). The purity of the RNA was tested using the quotient of the optical density (OD) at 260/280 nm. Then, the purified total RNA (1 mg) was reverse-transcribed using an EasyScript First-Strand cDNA synthesis super mix kit (TaKaRa, Dalian, China). The qPCR analysis was performed using SYBR Premix Ex Taq II. The PCR reaction was performed at 95 °C (5 min), followed by 45 cycles of denaturation for 95 °C (15 s), 60 °C (20 s), and 72 °C (20 s). The 2-ΔΔCt method was used to calculate the transcriptional levels of genes, with glyceraldehyde-3phosphate dehydrogenase (GAPDH) as the internal reference. Each sample was run in triplicate for qPCR. The sequences of primer pairs used in this assay were in the Table 1 .
Easyscript first strand cdna synthesis super mix kit
Manufactured by Takara Bio
Sourced in China, Japan
The EasyScript First-Strand cDNA synthesis super mix kit is a laboratory product designed for the reverse transcription of RNA into cDNA. This kit provides a convenient, one-step solution for the conversion of RNA into a DNA template suitable for downstream applications such as PCR amplification.
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Lab products found in correlation
2 protocols using easyscript first strand cdna synthesis super mix kit
1
Quantitative Analysis of Gene Expression in Rat Tissues
2
RNA Extraction and cDNA Synthesis
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Total RNA was extracted using RNAiso Plus (TaKaRa Biotechnology Co. Ltd., Dalian, China), strictly following the manufacturer instructions. The concentration of total RNA was checked using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Inc.) at 260 nmin terms of optical density values. The RNA integrality was assessed using 1.0% agarose gel electrophoresis. First-strand cDNA synthesis was performed from 4 µg of total RNA by using an EasyScript First-Strand cDNA Synthesis SuperMix kit (TaKaRa, Japan) following the manufacturer instructions. The reverse-transcription products were then used for polymerase chain reaction (PCR) amplification and stored at -80°C.
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