Total RNA was extracted from different tissues using a modified CTAB method, as previously described [37 (link)]. RNA extractions from petals at different flower developmental stages were carried out utilizing the Trizol method according to the manufacturer’s introduction (TaKaRa, Dalian, China). The first-strand cDNA was synthesized using the PrimerScript Reverse Kit (TaKaRa, Dalian, China). The real-time PCR primer pair for HcJMT1 was designed using Primer Premier 5 (Table S1 ), and its amplification specificity was confirmed via agarose gel electrophoresis and melting curve analysis. The amplification efficiency and correlation coefficient of the primer were determined using the standard curve method with a dilution series of cDNA templates. Real-time PCR was performed on an ABI 7500 Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA) using SYBR Premix Ex Taq (TaKaRa, Dalian, China), as described previously [37 (link),38 (link)]. Three independent amplifications were performed for each sample. Previously validated HcACT and HcRPS genes were used as reference genes for samples in different tissues and petals at different developmental stages, respectively [37 (link)]. The relative expression of HcJMT1 was calculated using the 2−ΔΔCt method [52 (link)]. Analysis of variance was conducted using SPSS 22 software with Tukey’s test (p = 0.05).
Primerscript reverse kit
Manufactured by Takara Bio
Sourced in China, Japan
The PrimerScript Reverse Kit is a reverse transcription kit that enables the conversion of RNA to cDNA. It contains a high-performance reverse transcriptase enzyme and necessary reagents to perform this reaction.
Automatically generated - may contain errors
2 protocols using primerscript reverse kit
1
Quantifying Gene Expression via qRT-PCR
2
Quantifying Omentin-1 mRNA Expression
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Total RNA isolation was undertaken by the use of TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) in keeping with the protocols put forward by the vendor. The reverse transcription into cDNA was carried out employing PrimerScript reverse kit (Takara, Dalian, Japan). The PCR reaction was conducted on the ABI Prism 7000 Sequence Detection System (ABI/Perkin Elmer, Foster City, CA, USA). The cycle conditions of PCR reaction were listed below: denaturation at 95°C for 20 s, 30 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s. The calculation of the mRNA expression of omentin-1 was verified by means of 2 -ΔΔCT method. Each assay was performed in triplicate.
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