Polyattract mrna kit

Leaf tissue from each time point and treatment was homogenized into liquid N₂. Total RNA was extracted using TRI-Reagent (Helena Bioscience) by following the manufacturer’s instructions. RNA was resuspended in 0.15% DEPC treated water and stored at -80°C. In order to remove any genomic DNA contamination, samples were treated with a DNase I enzyme kit (Invitrogen) following the manufacturer’s instructions. For RNA quantity and quality analyses, absorbance values at 260 nm and 280 nm (A₂₆₀, A₂₈₀) were determined. The concentration of RNA was calculated from A₂₆₀ values (A₂₆₀ = 1 corresponds to a concentration of 40 μg/ml). The ratio A₂₆₀/A₂₈₀ was used to assess total RNA purity. For the SSH library, 150 μg of total RNA was taken from each time point (50 μg/n x 3n = 150 μg) and mixed together (5 time points x 150 μg total RNA = 750 μg total RNA) so that each group (control and experimental) contained 750 μg of total RNA (tissue from 105 plants; 3 biological replications). mRNA was purified using a PolyATtract mRNA kit (Promega). mRNA precipitation was carried out overnight at -20°C in 0.1 volume of 3 M sodium acetate (pH 5.2) and 1 volume of isopropanol. For One-Step Real-Time Reverse Transcriptase-PCR (hereafter, qPCR) total RNA was extracted from rice tissue of each biological replication (n = 3; 7 plants/n) and each post-application time point (0.5-, 24-, 72 h).

Total RNA was isolated from 1 g of ground conserved tissue and extracted as described by Morcillo et al. (2006) [34 (link)]. RNA integrity was evaluated using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Before mRNA purification, 18 RNA samples of RRIM600 tissues and 12 extracts of RRIM600 OPS (open-pollinated seedlings) were pooled. Poly(A) RNA was isolated from these two pools with oligo beads (dT) from the PolyATtract mRNA kit (Promega, Madison, WI, USA).
Following isolation, the mRNAs were fragmented using a 0.1 M zinc chloride solution in 0.1 M Tris-HCl pH7.0. Using these shorter fragments as templates, the first-strand cDNAs were synthesized using Roche random primers and the AMV reverse transcriptase from the cDNA Synthesis system and the GS Rapid Library kits (Roche Applied Science, Mannheim, Germany). Sequencing was carried out on a Roche/454 GS-FLX (Titanium) pyrosequencing platform.