Nucleo spin plant rna isolation kit

Twenty selected genes that are differentially expressed have been further validated using qRT-PCR on the ABI 7500 system (Applied Biosystems, MA, USA) for the genotypes SS and ST. The names of the genes and the forward and reverse primers used for qRT-PCR are given in the Supplementary File S4. Total RNA was extracted from the seedlings treated with 200 mM sodium chloride using nucleo-spin plant RNA isolation kit (MACHERY-NAGEL) as per the instructions given. An amount of 3 µg of total RNA was translated into cDNA using a first-strand synthesis kit (Thermo Scientific, Waltham, MA, USA). SYBR Green Master Mix (2x, Takara, Shiga, Japan) was used as per the recommendations with the following thermal cycles: 1 cycle at 95 °C for 10 min, followed by 40 cycles alternatively at 95 °C for 15 s and 60 °C for a minute. After the 40th cycle, amplicon dissociation curves were recorded through raising the temperature from 58 to 95 °C within 20 min. Three biological replicates and two technical replicates were taken for analysis. Gene expression data were normalized with the sorghum genes Acyl Carrier Protein 2 (SbACP2) and Elongation Factor p (SbEF-p). Relative gene expressions were calculated through deploying REST software [121 (link)].

To examine the expression pattern of NRAT1 of the two rice varieties seedlings were exposed to AlCl₃ concentrations 0 and 100 μM for 24 and 48 hrs. Total RNA was extracted using Macherey-Nagel (Duren, Germany) Nucleospin plant RNA isolation kit following manufacturer’s instruction. One microgram of total RNA was used for first-strand cDNA synthesis using Revert Aid kit (Thermo Scientific, USA) following the manufacturer’s instructions. One-twentieth of the reaction volume was used as template for the PCR amplification of NRAT1 and Actin was used as an internal control (32 cycles). The primer sequences for semi quantitative RT-PCR of NRAT1 were 5′-GAGGCCGTCTGCAGGAGAGG-3′ and 5′-GGAAGTATCTGCAAGCAGCTCTGATGC-3′ [25 (link)] the forward and reverse sequences used to amplify Actin were 5’-ATGGCTGACGGCGAGGACATC-3’ and 5’-CAATACCATGCTCGATCGGGTA -3’, respectively.