Maxima h minus m mulv reverse transcriptase

One microgram of DNase treated total RNA was used for first strand cDNA synthesis using Maxima H Minus M-MuLV reverse transcriptase (Thermo Scientific, USA). A 5-fold dilution of cDNA samples was done with deionized water (Sigma, USA) to be used in qRT-PCR reactions. qRT-PCR was carried out using 2X Brilliant III SYBR® Green Q PCR (Agilent Technologies, USA) on a Stratagene Mx3000P (Agilent, USA) with cycling conditions and replications as described in Tiwari et al.^{20 (link)}. The amount of transcript accumulated for each target gene normalized to the reference gene Actin. The qRT- PCR primers were designed using the IDT Primer Quest software with default parameters: Tm = 59–62 °C; GC = 45–55%; amplicon size = 100–150 bp (Table S1). The heatmap for expression profiles of DEGs was generated using the MEV4 software package^{21 (link)}.

The total RNA from samples was isolated using RNAiso plus Reagent (TaKaRa, Japan) according to manufacturer’s instructions. RNA concentration of each sample was determined using NanoDrop- 1000 spectrophotometer (NanoDrop Technologies). The OD₂₆₀/OD₂₈₀ nm absorption ratio (1.98–2.01) and OD₂₆₀/OD₂₃₀ (≥2.0), was used to determine the quality and purity of RNA preparations. RNA integrity of each sample was also verified by electrophoresis in 1.2% formaldehyde-agarose gel in 1 × 3-(N-morpholino) propanesulfonic acid (MOPS) buffer prepared using diethyl pyrocarbonate (DEPC) treated water. Approximately 5 μg of total RNA was DNase treated to completely eliminate DNA contamination using RNase free DNase set (Qiagen, Germany). The first-strand cDNA synthesis was carried out using 1 μg of total Dnase free total RNA primed with oligodT primers in a final reaction volume of 20 μl using Maxima H Minus M-MuLV reverse transcriptase (Thermoscientific, USA) following manufacturer’s instructions and stored at −20 °C.