Reliaprep rna kit

RNA was extracted from leaves of four developmental stages, and from the two different cell types (NPCD and PCD cells). RNA was extracted from 40 mg of flash-frozen, midrib free leaf lamina tissue from one of each imperforate, pre-perforation, window or mature stage leaves from 3 different whole-plant cultures as per instructions for the ReliaPrep RNA Kit (Promega). RNA samples were treated with DNAse I (Thermo Fisher). Eluted RNA quantity was estimated using a Nanodrop spectrophotometer (Thermo Fischer) and a RNA integrity number (RIN) was determined using a Bioanalyzer (Agilent Technologies Inc., Santa Clara, CA, USA). Only RNA samples with a RIN ≥ 6.5 were approved for cDNA conversion.

HEK-293T cells were grown in DMEM supplemented with 10% foetal bovine serum and transiently transfected with plasmid DNA in the presence of Fugene-HD (Promega, Madison, WI, USA), as previously reported [21 (link)].
Upon transfection, cells were harvested by trypsinization, pelleted, washed twice with PBS, and aliquoted. A small aliquot (<0.5 × 10⁶ cells) was used for total RNA extraction using a Relia-Prep RNA kit (Promega), and the remaining cells (2–10 × 10⁶ cells) were resuspended in a 0.1 M Tris/HCl buffer, pH 7.5, containing 0.5% TritonX-100, vortexed to homogeneity, and stored in aliquots at −80 °C. After thawing on ice, the crude homogenate was used as the enzyme source for the in vitro assay. Protein content was determined by the bicinconic acid method (BCA protein kit, ThermoFischer Scientific). Homogenates for enzyme assay were kept at a protein concentration of about 10–20 mg/mL.
Human neonatal (P10857) and adult (P10858) fibroblasts were purchased from Innoprot (Bizkaia, Spain) and cultured in DMEM supplemented with 10% foetal bovine serum and antibiotics. Cell harvesting and aliquot preparation were performed, as was the case for transfected HEK-293 T cells.

RNA was extracted from individual leaf stages from plants grown for 30 d under normal conditions to measure the accumulation of Atg16 mRNA during lace plant leaf development. Mid-rib free leaf lamina tissue samples were washed with distilled water, blotted dry, and flash-frozen. RNA was extracted from 40 mg of frozen tissue from one of each imperforate, pre-perforation, window, or mature stage leaves from 3 different whole-plant cultures and processed as per instructions for the ReliaPrep RNA Kit (Promega). RNA samples were treated with DNAse I (Thermo Fisher). Eluted RNA was quantified using a Nanodrop spectrophotometer (Thermo Fischer) by measuring absorbance at 260 nm.
RNA was extracted from window and mature leaf stages from treated and control plants in the same manner as described above. Leaf lamina tissue samples were obtained from 3 different whole-plant cultures treated with either rapamycin, wortmannin, ConA, or DMSO (control).