Total RNA was isolated from the tissue samples using the Easy spin kit for total RNA extraction following the manufacturer’s instructions (INTRON Biotechnology, Korea). Nanodrop spectrophotometer (Genway Nanodrop, Germany) was used to determine RNA’s purities and concentrations. Using the RT-Premix Kit, 1 µg of RNA was used to produce cDNA (INTRON Biotechnology). A final volume of 20 µL was created by combining 2 µL of RT product with 10 µL of SYBR-Green master mix, 0.5 mM of each forward and reverse primer (Table 3 ), and nuclease-free water. All reactions were done at 95°C for 10 min, then 40 cycles of 95°C for 15 s, 58°C for 15 s, and 72°C for 30 s using a 7,500 Applied Biosystems, United States. The housekeeper gene β-actin was used to standardize the relative expression of mRNA. The 2−ΔΔCt approach, which Livak and Schmittgen (2001) (link) developed, was used to calculate the fold changes in mRNA expression.
Easy spin kit
Manufactured by iNtRON Biotechnology
The Easy Spin kit is a laboratory equipment designed for the rapid and efficient extraction and purification of nucleic acids, including DNA and RNA, from various biological samples. The kit utilizes a simple spin column-based method to isolate and concentrate target molecules, enabling users to obtain high-quality nucleic acid preparations for downstream applications.
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Lab products found in correlation
2 protocols using easy spin kit
1
Quantifying mRNA Expression via qRT-PCR
2
Ginseng Transcriptome Analysis by qRT-PCR
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Total RNA was isolated from 200 mg of ground ginseng leaves using the Easy Spin kit (iNtRON Biotechnology, Korea) according to the manufacturer's instructions. For the first strand complementary DNA (cDNA) synthesis, reverse transcription was performed using iScriptTM cDNA synthesis kit (Bio-Rad, Korea) according to the manufacturer's instructions. qRT-PCR was performed with the LightCycler system (Bio-Rad) using SYBR Green Sensimix Plus Master Mix (Bio-Rad) under the following conditions: 15 min at 95°C; 40 cycles of 30 s at 95°C, annealing for 30 s for the different primers at 60°C, 30 s at 72°C, and then 7 min at 72°C. Data were subjected to an analysis of variance (ANOVA) using Statistical Package for the Social Sciences (SPSS) statistics v. 18 (SPSS Inc., IBM, Armonk, NY, USA). The means were compared using Tukey's honest significant difference test at p < 0.05 to identify significant differences in levels of gene expression.
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