Computational protocol: Retinal Expression of Wnt-Pathway Mediated Genes in Low-Density Lipoprotein Receptor-Related Protein 5 (Lrp5) Knockout Mice

Similar protocols

Protocol publication

[…] Total retinal RNA was isolated from 3 Lrp5−/− and 3 WT mice. Gene expression microarray analysis was performed using an Illumina mouse gene microarray, with each sample being a biological replicate (n = 3 per group; Mouse-WG6 expression BeadChip; Illumina, San Diego, CA). All data is MIAME compliant and the raw data was deposited in a MIAME compliant database (GEO, accession number GSE32145). The chip contains ∼45,000 probe sets representing ∼34,000 genes. Microarray studies, from cDNA synthesis to raw data normalization were performed by the Molecular Genetics Core Facility at Children's Hospital Boston. Briefly, total RNA (1 µg each) were reverse transcribed, followed by a single in vitro transcription amplification to incorporate biotin-labeled nucleotide, and subsequent hybridization and staining with strepatavidin-Cy3 according to the manufacturer's instructions. The chip was scanned with Illumina BeadArray Reader to measure the signal intensity by the labeled target. Raw data were analyzed with the microarray software (Bead Studio Gene Expression version 3.4.0) for quality control, background analysis and normalization with rank invariant algorithm. Normalized data were further analyzed with the SAM program (Significant Analysis of Microarray) using p<0.05 and a delta of 0.19. Resulting gene lists for both Lrp5−/− and WT retinas were grouped using online tool Gene Ontology Enrichment Analysis Software (GOEAST Tools), which is hosted by Institute of Genetics and Developmental Biology, Chinese Academy of Sciences . Heat maps demonstrating differential gene expression were generated by adjusting the average signal of each sample to their respective log10 values. The average of the three WT signals was then used as the baseline for normal gene expression. Each value from Lrp5−/− retina was normalized to the average WT value and assigned a number between −1, which represented downregulation, 0, which represented no gene regulation, and +1, which represented upregulation. These values were then plotted in Microsoft Excel and colors were assigned to values using the Conditional Formatting function. Maps were then imported into Adobe Illustrator (CS4) to enhance contrast and resolution. [...] PCR primers targeting Lrp5 (F: 5′-AAG GGT GCT GTG TAC TGG AC-3′, R: 5-AGA AGA GAA CCT TAC GGG ACG-3′), Frizzled4 (F: 5′-TTC CTT TGT TCG GTT TAT GTG CC-3′, R: 5′-CTC TCA GGA CTG GTT CAC AGC-3′), Norrin (F: 5′-GTG AGG GGC ACT GCA GCC AG-3′; R: 5′-CAG CGC AGA CGC AGA GCC TT-3′), Claudin5 (F: 5′-GCA AGG TGT ATG AAT CTG TGC T-3′, R: 5′-GTC AAG GTA ACA AAG AGT GCC A-3′), Plvap (F: 5′-GCT GGT ACT ACC TGC GCT ATT-3′, R: 5′-CCT GTG AGG CAG ATA GTC CA-3′), Wnt5a (F: 5′-CAA CTG GCA GGA CTT TCT CAA-3′, R: 5′-CAT CTC CGA TGC CGG AAC T-3′), Wnt7b (F: 5′-GGT GTG GCA GTG TAC CTG CAA-3′, R: 5′-GTG AAG ACC TCG GTG CGC T-3′), Wnt10b (F: 5′-GAAGGGTAGTGGTGAGCAAGA-3′, R: 5′-GGT TAC AGC CAC CCC ATT CC-3′), Gja1 (F: 5′- ACA GCG GTT GAG TCA GCT TG-3′, R: 5′- GAG AGA TGG GGA AGG ACT TGT -3′), EMP1 (F: 5′- TTG GTG CTA CTG GCT GGT CT -3′, R: 5′- CAT TGC CGT AGG ACA GGG AG-3′), Mfsd2 (F: 5′- AGA AGC AGC AAC TGT CCA TTT -3′, R: 5′- CTC GGC CCA CAA AAA GGA TAA T-3′), Sox18 (F: 5′- ATG CCA CTA CAC TCC CCT ACC A-3′, R: 5′- CTG CTC TCT TCT GGA CAG GAC AT-3′), slc38a5 (F: 5′- CAA CCT CAG CAA CGC TAT CAT-3′, R: 5′- CAG GTC CAA ATG CCC TCT G-3′), Dvl1 (F: 5′- ATG AGG AGG ACA ATA CGA GCC-3′, R: 5′- GCT TCC GAA CTA GCC GAG AG-3′), Dvl2 (F: 5′- TGT CGT CAG ATA CCC CAC AG-3′, R: 5′- CTG GAT ACA TTA GGG TGG AAG GA-3′), Dvl3 (F: 5′- ACA CGG AGA CCG ACT CCT T-3′, R: 5′- AGG GTA GAT GAA CTG TCA TAG CC-3′), vWF (F: 5′- CAA TGG CAC CGT AAC GCA G-3′, R: 5′- TGG AGA GCT TAT AGT ACC CAG C-3′) and an house keeping control gene, Cyclophilin A (F: 5′-AGG TGG AGA GCA CCA AGA CAG A-3′, R: 5′-TGC CGG AGT CGA CAA TGA T-3′), were designed using Primer Bank and NCBI Primer Blast Software.Express software (Applied BioSystems). We used three methods to analyze primer sequences for specificity of gene detection. First, NCBI Blast module was used to identify primer and probe sequences that specifically detected the sequence of choice. Second, amplicons generated during a PCR reaction were analyzed using the first derivative primer melting curve software supplied by Applied BioSystems. This analysis determines the presence of amplicons on the basis of a specific melting point temperature. Third, amplicons generated were gel purified and sequenced by the Children's Hospital Boston Core Sequencing Facility. This further confirmed the selection of the desired sequence. Quantitative analysis of gene expression was determined using an ABI Prism 7700 Sequence Detection System (TaqMan) and the SYBR Green Master mix kit. Standard curves for each gene were plotted with quantified cDNA template during each real-time PCR reaction. Each target gene cDNA copy number was normalized to 106 copies of the house keeping gene, cyclophilin A using delta-delta CT method. […]

Pipeline specifications

Software tools GOEAST, Adobe Illustrator, Primer-BLAST, BLASTN
Applications Miscellaneous, Gene expression microarray analysis, qPCR
Organisms Mus musculus, Homo sapiens
Diseases Eye Diseases, Retinitis