Total RNA was extracted using RNA extraction kit (9769; TaKaRa, Beijing, China) according to the manufacturer’s instructions. First-strand cDNA synthesis was performed using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (RR047A; TaKaRa, Beijing, China). The quality of the RNA samples were tested using NanoDrop2000c (Thermo Fisher Scientific, Waltham, MA, USA) and gel electrophoresis. Quantitative real-time PCR (qRT-PCR) was carried out using TB Green Premix Ex Taq II (6210A; TaKaRa, Beijing, China) on an FTC-3000P system (Funglyn Biotech, Toronto, Canada). Design the primer used Primer Premier 5.0 with the primers listed in Table S2 . The PCR conditions consisted of denaturation at 95 °C for 30 s, followed by 40 cycles of 5 s at 95 °C, and 30 s at 60 °C, and a final step at 4 °C. All samples were tested with three technical replicates and three independent biological replicates. The relative expression level was calculated using the 2−ΔΔCT method with yam ribosomal RNA (18S) as the internal control (internal reference genes screened by our laboratory) (Shao et al., 2021 (link)).
Transscript one step gdna removal and cdna synthesis supermix
Manufactured by Takara Bio
Sourced in China
TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix is a reagent designed for the simultaneous removal of genomic DNA and synthesis of complementary DNA (cDNA) from RNA samples. It provides a one-step solution for these two essential steps in RNA-based analyses.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000c, by Thermo Fisher Scientific (1 mentions)
Tb green premix ex taq 2, by Takara Bio (1 mentions)
Rna extraction kit, by Takara Bio (1 mentions)
Trizol reagent, by Takara Bio (1 mentions)
Tb green premix ex taq 2 tli rnaseh plus, by Takara Bio (1 mentions)
Abi 7500 fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
2 protocols using transscript one step gdna removal and cdna synthesis supermix
1
Extraction and Quantification of Yam RNA
2
RNA Extraction and Quantitative RT-PCR
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Total RNA from each sample was extracted with Trizol reagent (Takara). Quality of total RNA was determined by using agarose gel electrophoresis and nano drop nd-1000 full wavelength spectrophotometer. About 2 µg of total RNA was reverse transcribed into cDNA using cDNA rst strand synthesis kit (TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix) (TaKaRa) in a 20 µL reaction. Reverse transcription PCR (RT-PCR) was conducted by 35 cycles, with 18S rRNA as internal controls for H. ammodendron. Quantitive real-time PCR (qRT-PCR) was performed with an ABI 7500 fast real-time PCR system (Applied Biosystems). qRT-PCR reaction was performed using TB Green® Premix Ex Taq™ II (Tli RNaseH Plus) (TaKaRa) according to the manufacture instructions. 18S rRNA gene was used as the internal reference gene (Wang et al. 2018) (link), primers used are listed in Supplementary Table 1. The relative expression levels were calculated using the relative 2 -△△CT method (Livak and Schmittgen 2001) in three independent biological samples with three technical replicates.
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