Illustra mrna purification kit

Nematodes were cultured on Escherichia coli strain OP50 on NGM agar plates [20] (link). RNA was extracted from 580 mg (for a culture grown at 20°C, called PDT) and 730 mg (for a culture grown at 20°C and subsequently brought down to 4°C, called PDF) of P. davidi CB1 (wet weights), respectively. Total RNA (930 µg for PDT and 750 µg for PDF) was prepared with RNagents total RNA Isolation Kit (Promega). Poly(A)+ RNA (2.9 µg for PDT and 5.5 µg for PDF) were isolated with Illustra mRNA Purification Kit (GE healthcare). 2 µg of poly(A)+ RNA was then used to generate a cDNA library with Cloneminer cDNA library Construction Kit (GE healthcare).

Microarray analyses were conducted as described previously [44 (link)]. Maize internode sections were ground under liquid nitrogen and total RNA extracted with the TRIZOL reagent (Invitrogen, Sydney) according to the manufacturer’s instructions. Polyadenylated mRNA was purified using the Illustra mRNA Purification Kit (GE Biosciences). Sample quality and RNA concentration were assessed using an Agilent Bioanalyzer. The mRNA was reverse-transcribed into double-stranded cDNA, which was labelled with Cy3 or Cy5 fluorescent dye, using the Agilent Low RNA Linear Amp kit. Biological replicates were labelled alternately using Cy3 or Cy5 to guard against dye-bias. The cDNA was hybridized to Agilent 4 × 44 k maize gene microarrays [70 (link)] and the microarrays were washed according to Agilent standard protocols. The microarray chips were scanned with an Agilent G2505B DNA Microarray Scanner at two laser power settings (100% and 10%). The images were inspected visually for image artefacts, and feature intensities were extracted, filtered and normalized with Agilent Feature Extraction Software (v 9.5.1). The data were normalized using quantile normalization (BOLSTAD http://www.ncbi.nlm.nih.gov/pubmed/12538238). The normalized data were used to compare expression levels of genes related to cell wall compositions.

Total RNA was extracted from developing seeds (between 14 d and 18 d after pollination) of C. pulcherrima and C. viscosissima using a method as described (Suzuki et al., 2004 (link)) without 8M LiCl treatment. mRNAs were purified from ~1mg of total RNA by two passes through oligo(dT)–cellulose columns by use of the Illustra mRNA purification kit (GE Healthcare, Pittsburgh, PA, USA). A sequencing library optimized for Roche/454 GS FLX Titanium sequencing was prepared from oligo(dT)-enriched mRNA according to custom protocols used previously (Nguyen et al., 2013 (link)). To reduce the number of high copy transcripts, amplified dsDNA library intermediates were partially normalized using Trimmer Direct (Evrogen) protocols. Emulsion PCR and sequencing were performed according to the manufacturer (Roche/454 Life Sciences). High quality sequence reads were trimmed (https://sourceforge.net/projects/estclean/) and assembled using Newbler v2.0.