The reliability of the RNA-seq data was validated by qRT-PCR. The total RNA of 20 candidate genes was extracted using the CTAB method and an E.Z.N.A. Plant RNA Kit (Omega Bio-Tek), with three biological replicates per treatment. The first-strand cDNA was synthesised using EasyScript One-Step gDNA Removal and cDNA Synthesis Super Mix kit (Beijing TransGen Biotech Co., Ltd., China). The primers were designed using the Primer-BLAST (www.ncbi.nlm.nih.gov/tools/primer-blast/ ) tool from the National Centre for Biotechnology Information database (Supplementary Table S3 ). With the 18S rRNA gene as the reference control, the expression of each gene relative to the 18S rRNA gene was calculated using the 2-△△Ct method. The PCR program was as follows: step 1 (pre-denaturation): 95 °C, 2 min; step 2 (PCR amplification; 45 cycles): 95 °C, 15 s; Tm value, 15 s; 55 °C, 20 s; and step 3 (melting curve): 95 °C, 15 s; 60 °C, 15 s; δ, 20 min; and 95 °C, 15 s. The fluorescent dye used for the reactions was TB GreenTM Premix Ex TaqTM II (Tli RNase H Plus) RR820A kit (TaKaRa, Japan). The qRT-PCR was run in an Eppendorf Realplex 2 fluorescent quantitative PCR system (Eppendorf, Germany).
Tb green premix ex taqtm 2 tli rnase h plus rr820a kit
Manufactured by Takara Bio
Sourced in Japan
TB Green™ Premix Ex Taq™ II (Tli RNase H Plus) RR820A kit is a real-time PCR reagent that enables efficient amplification and detection of DNA sequences. The kit includes a DNA polymerase, buffer, and dye for real-time PCR applications.
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2 protocols using tb green premix ex taqtm 2 tli rnase h plus rr820a kit
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Validating RNA-seq Data by qRT-PCR
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Validation of RNA-seq Data by qRT-PCR
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The reliability of the RNA-seq data was validated by qRT-PCR. The total RNA of 20 candidate genes was extracted using the CTAB method and an E.Z.N.A. Plant RNA Kit (Omega Bio-Tek), with three biological replicates per treatment. The rststrand cDNA was synthesised using EasyScript One-Step gDNA Removal and cDNA Synthesis Super Mix kit (Beijing TransGen Biotech Co., Ltd., China). The primers were designed using the Primer-BLAST (www.ncbi.nlm.nih.gov/tools/primer-blast/) tool from the National Centre for Biotechnology Information database (Table S3). With the 18S rRNA gene as the reference control, the expression of each gene relative to the 18S rRNA gene was calculated using the 2 -△△Ct method. The PCR program was as follows: step 1 (pre-denaturation): 95°C, 2 min; step 2 (PCR ampli cation; 45 cycles): 95°C, 15 sec; T m value, 15 sec; 55°C, 20 sec; and step 3 (melting curve): 95°C, 15 sec; 60°C, 15 sec; δ, 20 min; and 95°C, 15 sec. The uorescent dye used for the reactions was TB GreenTM Premix Ex TaqTM II (Tli RNase H Plus) RR820A kit (TaKaRa, Japan). The qRT-PCR was run in an Eppendorf Realplex 2 uorescent quantitative PCR system (Eppendorf, Germany).
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