Computational protocol: Transcriptome profiling of Brassica napus stem sections in relation to differences in lignin content

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[…] Total RNA from stem sections of both DH and YN were extracted as described in Carpenter and Simon [] followed by clean-up using the commercial RNeasy mini kit (Qiagen, Valencia, CA, USA). Two biological replicates were collected for each sample. RNA amplification, labeling with cy3- or cy5-dCTP dyes (GE Healthcare, Buckinghamshire, UK), and probe fragmentation was carried out using Ambion AminoAllyl MessageAmp II RNA amplification kit according to the manufacturer’s instructions (Ambion, Austin, TX, USA). A spotted 15 K 50-mer B. napus oligo array, previously developed at the Saskatoon Research and Development Centre, Agriculture and Agri-Food Canada [], was hybridized with the cy5- and cy3-labelled probe pairs. Cy3 dye was used for labeling the YN RNA and Cy5 for DH RNA. A dye swap (cy3/cy5) labelling experiment was performed for each biological replicate. Labeling, hybridization and post- hybridization washing were conducted according to the protocol for Corning epoxide slides (Corning Inc., Lowell, MA, USA). Hybridization was carried out at 37 °C for 17 h in a MAUI hybridization station (BioMicro Systems, Salt Lake City, UT, USA). Following the post-hybridization washes, slides were scanned with a VersArray ChipReader laser scanner (Bio-Rad Laboratories, Hercules, CA, USA).Image files were transferred to ArrayPro Analyzer software (Media Cybernetics Inc., Bethesda, MD, USA) for image analysis, spot verification, normalization, filtering and feature extractions. A standard statistical program GeneSpring GX (Agilent Technologies, Santa Clara, CA, USA) was used to determine that the data set would fit a normal distribution (log 10) and to check the validity of expression data by assessing the variation between control spots. Data files for the duplicates on individual slides and the dye swap data files for each experiment were merged and average spot intensities used to reduce experimental bias. Normalization was performed on merged data using the Lowess (sub-grid) method, and the local background was subtracted from the values of each spot. The intensity of each spot at λ549 nm (Cy5) and λ647 nm (Cy3) was analyzed using a BASE plug-in and finally transformed into a DH/YN ratio value that denoted the most upper (section 1) and lowest stem sections (section 4), including DH1:YN1, DH4:YN4, DH1:DH4 and YN1:YN4. Additional data processing was performed using tools available in BASE (http://base.thep.lu.se). Gene ontology (GO) analysis was conducted using the TAIR database []. Microarray ratios with transcript changes > 2-fold upregulated or downregulated between two different B. napus stem sections were considered for all ratios where p ≤ 0.05 (Additional files , , , , , , , , , , and : Table S1A and B to S6A and B). Data in these 12 Additional files were screened using either Excel or Microsoft Access to identify individual or groups of B. napus genes expressed in multiple DH and YN ratios in the main categories of cell wall, other carbohydrate genes and transcription factors, and in additional subcategories. Ten major transcription factor families, Arabidopsis homologue IDs and corresponding mutant lines for selected B. napus genes were retrieved from http://www.arabidopsis.org/browse/genefamily/index.jsp []. [...] For quantitative real time reverse transcription PCR (qRT-PCR) experiments, total RNA was extracted from stem sections using a TRIzol reagent (http://www.invitrogen.com) following the manufacturer’s instructions. Three biological replicates (independent RNA preparations) were collected and used with three technical reps for qRT-PCR analysis. RNA concentration was determined using a nanodrop spectrophotometer (Fisher Scientific, Canada). Twenty micrograms of total RNA (per sample in each replicate) was treated with Turbo DNase (www.ambion.com) as per the manufacturer’s instructions to eliminate trace amounts of genomic DNA. Reverse transcription reactions were performed with Superscript™ III Reverse Transcriptase (http://www.invitrogen.com) according to the manufacturer’s instructions using 1.5 μg RNA per reaction. Two RT reactions per sample were done and reactions were pooled after 10-fold dilution with nuclease-free water. Polymerase chain reactions were carried out using 96-well plates in a LightCycler® 480 II (http://www.roche-applied-science.com/lightcycler-online) using SYBR® Green. Reactions contained 10 μl of 2× SYBR® Green Master Mix (Roche), 5 μl of diluted cDNA and 200 nM of gene/EST-specific primer (Additional file : Table S7) in a final volume of 20 μl. For data normalization, five reference genes (actin, adenine phosphorybosyl transferase, β-tubulin, cyclophilin and elongation factor 1-α) were included in the experiment and two genes with stable expression, namely β-tubulin and actin, were selected using geNorm software []. PCR efficiency, in the range of 85 to 100%, was determined from amplification plots using the program LinRegPCR []. […]

Pipeline specifications

Software tools GeneSpring GX, LinRegPCR
Applications Gene expression microarray analysis, qPCR
Organisms Brassica napus, Homo sapiens