Computational protocol: A Lectin-Based Glycomic Approach to Identify Characteristic Features of Xenopus Embryogenesis

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Protocol publication

[…] The design and use of the lectin microarray was described previously () , . Briefly, triplicate probes of 96 lectins at 0.5 mg/mL were spotted onto glass slides. Embryo samples were homogenized in modified RIPA buffer , and clarified supernatants were obtained after centrifugation using a conventional protein extraction procedure. The extracts were labeled with Cy3-N-hydroxysuccinimide (NHS) ester (GE Healthcare), diluted to 0.5 µg/ml, and incubated with the lectin microarray at 20°C overnight. Fluorescence images were acquired using an evanescent-field activated fluorescence scanner (GlycoStation Reader 1200; GP Biosciences). The fluorescence signal of each spot was quantified using an Array-Pro Analyzer ver.4.5 (Media Cybernetics), and a background value obtained from an area without lectin immobilization was subtracted from each spot. Lectin signals from triplicate spots were averaged and normalized to the mean value of the 96 lectins in the array.Unsupervised clustering was performed by the average linkage method using Cluster 3.0 software. The heat map with clustering was obtained using Java TreeView. Differences between 2 arbitrary data sets were evaluated by applying Student's t-test to each lectin signal using SPSS statistics 19 (SPSS). [...] Total RNA was extracted from frozen samples of each embryonic stage using ISOGEN (NIPPON GENE) according to the manufacturer's instructions. Samples were analyzed by Agilent Frog Microarray 4×44 k (G2519F, Agilent Microarray Design ID 015066) using the Quick-Amp Labeling Kit, one color (Agilent). Arrays were scanned using a G2505C Microarray Scanner System (Agilent). Raw microarray data were submitted to the Gene Expression Omnibus (GEO) microarray data archive (http://www.ncbi.nlm.nih.gov/geo/) at the NCBI (accession numbers GSE40620).Data were analyzed using Gene-Spring GX12.0 software (Agilent) after applying 2 normalization procedures: (1) signal intensities of <1 were set to 1, and (2) each chip was normalized to the 75th percentile of all measurements from that chip. Baseline transformation of those data was not performed. Genes with a flag value of “detected” or “not detected” in at least 1 sample were analyzed. “Xenopus glycogenes” gene sets are listed in (see text). A heat map in which each square element indicates the correlation coefficient value between 2 developmental stages was acquired using matplotlib (http://matplotlib.sourceforge.net/). […]

Pipeline specifications

Software tools TreeView, SPSS, matplotlib
Databases GEO
Applications Miscellaneous, Gene expression microarray analysis
Organisms Xenopus laevis
Diseases Neoplasms