Adult worms were collected from infected pigs, and fresh tissues were dissected and snap frozen in liquid nitrogen for subsequent storage at -80 °C. Total RNA of each individual tissue was prepared using TRIzol (Invitrogen) according to the manufacturer’s instruction and treated with Ambion Turbo DNase (Ambion/Applied Biosystems, Austin, TX). RNA quality and quantity were assessed by Agilent 2100 Bioanalyzer (Agilent Technologies, Cedar Creek, Texas), and the yield determined using the NanoDrop ND-1000 UV-VIS spectrophotometer v.3.2.1 (NanoDrop Technologies, Wilmington, DE).
Fluorescence labeled cDNA was prepared from ~10 ug of total RNA using Superscript Plus Indirect labeling system (Invitrogen), with slight modifications. In brief, The anchored oligo(dT)20 primer is a mixture of 12 primers, each consisting of a string of 20 deoxythymidylic acid (dT) residues followed by two additional nucleotides represented by VN, where V is dA, dC, or dG, and N is dA, dC, dG, or dT. The VN “anchor” allows the primer to anneal preferentially at the 5´ end of the poly(A) tail of mRNA, ensuring high specificity, high yields, and longer lengths of cDNA. Two aminoallyl-modified nucleotides were used together with other dNTPs in reverse transcription, for incorporation into the cDNA. After RNA hydrolysis, neutralization, and column purification, the amino-modified cDNA is coupled with a monoreactive, N-hydroxysuccinimide (NHS)-ester fluorescent dye, either Alexa Fluor 555 succinimidyl ester or Alexa Fluor 647 succinimidyl ester. The Alexa dyes provide higher correlation coefficients, signal intensities, and single-to-background ratios. After the final column purification, the fluorescence labeled single-strand cDNA samples are quantified via spectrophotometer.
species: Ascaris suum tissue: Pooled tissues gender: Pooled genders
Extracted molecule
total RNA
Extraction protocol
Adult worms were collected from infected pigs, and fresh tissues were dissected and snap frozen in liquid nitrogen for subsequent storage at -80 °C. Total RNA of each individual tissue was prepared using TRIzol (Invitrogen) according to the manufacturer’s instruction and treated with Ambion Turbo DNase (Ambion/Applied Biosystems, Austin, TX). RNA quality and quantity were assessed by Agilent 2100 Bioanalyzer (Agilent Technologies, Cedar Creek, Texas), and the yield determined using the NanoDrop ND-1000 UV-VIS spectrophotometer v.3.2.1 (NanoDrop Technologies, Wilmington, DE).
Fluorescence labeled cDNA was prepared from ~10 ug of total RNA using Superscript Plus Indirect labeling system (Invitrogen), with slight modifications. In brief, The anchored oligo(dT)20 primer is a mixture of 12 primers, each consisting of a string of 20 deoxythymidylic acid (dT) residues followed by two additional nucleotides represented by VN, where V is dA, dC, or dG, and N is dA, dC, dG, or dT. The VN “anchor” allows the primer to anneal preferentially at the 5´ end of the poly(A) tail of mRNA, ensuring high specificity, high yields, and longer lengths of cDNA. Two aminoallyl-modified nucleotides were used together with other dNTPs in reverse transcription, for incorporation into the cDNA. After RNA hydrolysis, neutralization, and column purification, the amino-modified cDNA is coupled with a monoreactive, N-hydroxysuccinimide (NHS)-ester fluorescent dye, either Alexa Fluor 555 succinimidyl ester or Alexa Fluor 647 succinimidyl ester. The Alexa dyes provide higher correlation coefficients, signal intensities, and single-to-background ratios. After the final column purification, the fluorescence labeled single-strand cDNA samples are quantified via spectrophotometer.
Hybridization protocol
Each cDNA sample was paired and balanced with the common reference, and applied to a single plex of the custom array. Hybridization was carried out at 65 ºC for 20 hours. Washing procedures were carried out according to the Agilent protocols.
Scan protocol
Slides were scanned with an Axon 4000B scanner for the detection of fluorescent signals. Laser power was kept constant and PMT was varied for each slide to ensure optimal signal intensities while maintaining saturation below 0.1%. Images were processed using GenePix v6.1 (Axon) to generate gpr files containing raw intensities, gene information, etc.
Data processing
Scanned GPR files were first inspected for quality control (QC) using built-in QC tools from the Bioconductor package of R. Inspected microarrays were then analyzed using LIMMA of Bioconductor. After background correction and normalization, gene expression was detected through a linear model fitting which considers both the dye effect and the variation among replications. Differentially expressed genes were defined based on the F test of the linear model fitting. To control the problem of multiple tests, we used false discovery rate (FDR) of 0.01 as a cutoff. The resulting differentially expressed gene set with corrected F-test and FDR was exported for further analysis.
Gene over- and under-expression was determined using the “2 fold” rule based on the linear modeling fitting results. For each gene, if the fitted effect coefficient for one tissue was more than 1 (log-ratio compared with the signal of pooled references), it was recorded as relatively over-expressed in this tissue; if the coefficient was less than -1, it was recorded as relatively under-expressed in that tissue. By this way, each tissue was assessed for its complement of over or under- expressed genes. We further examined the functions of predicted proteins encoded by the genes that were exclusively over-expressed in a specific tissue by GO term enrichment analysis using the Func program.
