Agilent 2100 bioanalyzer rna chip

Four tissues of C. oleifera, including tender shoots, young leaves, flower buds and flowers, were harvested in 2010 from East Park of Kunming Botanic Garden, Yunnan Province, China. All necessary permits were obtained from Wei-bang Sun, who is the director of the Kunming Botanical Garden, the Chinese Academy of Sciences. All samples were flash frozen in liquid nitrogen and stored at −80°C for RNA extraction. Total RNA was extracted by a modified CTAB method. The quality and quantity of total RNA were analyzed using agarose gel electrophoresis and Agilent 2100 Bioanalyzer RNA chip (Agilent Technologies, CA). cDNA library construction and normalization were performed as described previously [44] (link). All libraries were combined into a single pool and sequenced using the 454 GS-FLX platform (Roche, IN, USA).

Frozen tissues were ground to a fine powder in liquid nitrogen and total RNA was extracted using the RNeasy plant Mini Kit (QIAGEN, Germantown, MD—USA) and treated with RNase free DNaseI (QIAGEN) according to the manufacturer’s instructions. The quality and quantity of total RNA were analyzed using the Agilent 2100 Bioanalyzer RNA chip (Agilent Technologies Inc., Santa Clara, CA—USA). The RNA samples with an RNA integrity number (RIN) higher than 7.0 were selected and used for subsequent analyses.

Fruits were sampled at the last two ripening stages, during maturation of the pericarp, around 210 days after flowering (DAF) (hereafter ‘yellow stage’) (stage 6) and around 240 DAF (hereafter ‘red stage’) (stage 7). Each of the three biological replicates contained beans from a different tree. Twenty beans were sampled from different sides of the canopy of each tree. Representative portions of the fruit endosperm tissues were frozen in liquid nitrogen and stored at − 80°C until use. Total RNA was extracted by grinding 100 mg aliquots of frozen tissues to a fine powder in liquid nitrogen using the Qiagen "RNeasy® Lipid Tissue" kit (QIAGEN, Germantown, MD, USA). The quality and quantity of total RNA were estimated using an Agilent 2100 Bioanalyzer RNA chip (Agilent Technologies Inc., Santa Clara, CA—USA). The RNA samples with RNA integrity number (RIN) higher than 7.0 were selected and used for the subsequent analysis.

Callus sample of selected stages with at least three independent sample of each stage were harvested, ground in liquid N₂ and stored at −80 °C. Frozen tissues were ground to a fine powder in liquid nitrogen and total RNA was extracted using RNeasy plant Mini Kit (QIAGEN, MD) and treated with RNase free DNaseI (QIAGEN, MD) according to manufacturer’s instructions. The quality and quantity of total RNA were analyzed by agarose gel and spectrophotometric analysis (ND1000 Nanodrop, NanoDrop Technologies, USA). Quantity as well as quality of pooled RNA was again checked using Agilent 2100 Bioanalyzer RNA chip (Agilent Technologies Inc., Santa Clara, CA). Only the RNA samples with 260 of 280 ratios from 1.8 to 1.9, 260 of 230 ratios from 2.0 to 2.5 and RIN (RNA integrity number) more than 9.0 were used for the analysis. The equal amount of total RNA from the three independent samples of each stage were pooled and used for further processing.

Total RNA was extracted from three parallel independent biological sets of unwounded and 20 min wounded leaves using plant total RNA isolation kit (Sigma) according to manufacturer’s instructions. The concentration of RNA samples was determined using a Nanodrop ND-1000 spectrophotometer (Nanodrop technologies, Wilmington, DE, USA). The quality and quantity of RNA was again checked using Agilent 2100 Bioanalyzer RNA chip (Agilent Technologies Inc., Santa Clara, CA). The integrity of RNA was assessed by electrophoresis on a 1.2% agarose gel in 0.5X TBE. Only the RNA samples with 260/280 ratios from 1.8 to 1.9, 260/230 ratios from 2.0 to 2.5 and RIN (RNA integrity number) more than 6.0 were used for the analysis. This RNA was used for Illumina sequencing and gene expression analysis.