Sv total rna isolation system extraction kit

Tissue samples collected for the analysis of the expression of NF-ĸB p65,TNF-α, IL-6 gene involved in the inflammatory process were stored in an RNA Shield solution at a temperature of −80 °C until processing. The total RNA was extracted using an SV Total RNA Isolation System extraction kit, purchased from Promega, according to the manufacturer’s recommendations. The quantitative and qualitative of purified RNA evaluation was assessed spectrophotometrically, using the NanoDrop 8000 spectrophotometer produced by Thermo Scientific, Waltham, MA, USA. The conversion of the total RNA to complementary DNA was performed using 2 micrograms of total RNA and the First Strand cDNA Synthesis Kit conversion kit. To determine the quantitative expression, we used a LuminarisHiGreenqPCT Master Mix kit (Thermo Scientific, Waltham, MA, USA), low ROX, each sample being determined in triplicate. The PCR system used was Applied Biosystems 7500 Real Time PCR System (Foster City, CA, USA). Primers used were included in Table 1. The results obtained were interpreted using the 2ΔΔCT method, proposed by Livak in 2001 [34 (link)].

For the total RNA extraction from the roots of vanilla plants, a protocol was standardized based on a previous report [80 (link)]. Briefly, 200 mg of root tissue were homogenized with the Trizol reagent and then treated with Phenol:Chloroform:Isoamyl Alcohol (25:24:1), followed by vortexing and centrifugation. The upper aqueous phase was transferred into silica columns included in the SV Total RNA Isolation System extraction kit from Promega. The integrity of the obtained RNA was determined by electrophoresis in 2% agarose gel, stained with ethidium bromide (EtBr 0.5 μg ml^− 1) under denaturing conditions. The concentration of total RNA samples was verified using a NanoDrop spectrophotometer, as well as its RNA Integrity Number (RIN) values were obtained with an Agilent 2100 Bioanalyzer system (Agilent Technologies). RNA samples with RIN values > 6 were used for cDNA synthesis and subsequent sequencing.