Low input quick amp labeling kit

Labeling of isolated RNA was done using the Low Input Quick Amp Labeling kit (Agilent Technologies, Palo Alto, CA, USA), according to the manufacturer’s instructions. For whole-genome expression analysis, samples were hybridized to Agilent 4x44K v2 Whole Human Genome arrays (G4845A; Agilent Technologies), covering 27,958 genes. In brief, equal amounts of total RNA (50 ng) were amplified and labeled with either Cy3-CTP (experimental samples) or Cy5-CTP (reference material, obtained as described above) using the Low Input Quick Amp Labeling kit (Agilent Technologies). For hybridization, equal amounts (825 ng) of labeled samples were fragmented in Fragmentation Buffer (Agilent Technologies) for 30 min at 60 °C. Labeled and fragmented complementary RNA (cRNA) was hybridized to the array and incubated in a rotating hybridization chamber for 17 h at 60 °C. After hybridization, the array was washed subsequently for 5 min in 6 x saline sodium phosphate-EDTA (SSPE)/0.005% N-lauroylsarcosine, 1 min in 0.006 x SSPE/0.005% N-lauroylsarcosine, and 30 s in acetonitrile and dried quickly in nitrogen flow. The arrays were scanned at a resolution of 5 mm and at 5 and 100% photomultiplier tube settings using the Agilent DNA Microarray Scanner (Agilent Technologies). Scan data were extracted using Agilent Feature Extraction software (version 8.5.1; Agilent Technologies).

Eleven Ftx^–/–, three Ftx^–/Y, four WT female and three WT male eyes were analysed. Forty-two duplicate hybridization experiments were carried out. In E13.5 mouse embryos, eyes from which the pigmented epithelial layer had been removed surgically were collected for analysis. Total RNA was extracted from each eye sample using RNeasy micro kits (QIAGEN, Germany). Eluted RNA was quantified using an Agilent 2100 Bioanalyzer instrument, and 200 ng was used for amplification and labelling using Low Input Quick Amp Labeling kits (#5190-0442, Agilent Technologies, USA) following the manufacturer’s instructions. Cy3-labelled cRNA was purified using RNeasy mini spin columns. The amount of labelled cRNA and uptake of cyanine were measured using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific, USA). The labelled probes were hybridized to an Agilent 8 × 60 K whole-mouse genome microarray (G4852A). After washing, microarrays were scanned using an Agilent Microarray scanner. The data were normalized using the R statistical package with the “qspline” function of the “affy” package in Bioconductor software (https://www.bioconductor.org/install/).

Purified total RNA (200 ng) was reverse-transcribed to cDNA and then amplified to Cy5-labeled cRNA with Cy5-CTP-labeled nucleotides (Perkin Elmer) as previously described [78 (link)] using Low Input Quick Amp Labeling kits (Agilent), as per manufacturer’s instructions for hybridization to a 4-pack oligo gene expression microarray. Labelled cRNA was purified through RNeasy columns as per manufacturer’s instructions (QIAGEN) and quantified using spectrophotometry (NanoDrop-1000), ensuring specific activity of all samples > 6 pmol dye per microgram cRNA (Agilent). Samples were kept at −80 °C until hybridization. A reference pool of Cy3-cRNA was synthesized by amplifying experimental samples as described above, but with Cy3-CTP-labeled nucleotides (Perkin Elmer). In each experiment, a reference pool of equimolar cRNA was generated from each experimental condition (n = 10 individuals).