Genechip scanner 3000 7g system

Total RNA was isolated and purified from conjunctival epithelium using the miRNeasy mini kit (Qiagen, Tokyo, Japan) for microarray profiling of miRNAs as recommended by the manufacturer. For all samples the RNA integrity number (RIN) was > 7. Affymetrix GeneChip miRNA 4.0 arrays were used for miRNA profiling. As described by the manufacturer, total RNA was labeled using the FlashTag Biotin HSR RNA labeling kit (Affymetrix Inc., USA). The samples were hybridized on GeneChip miRNA 4.0 arrays (Affymetrix) for 18 h at 48 °C. The arrays were then washed to remove non-specifically bound nucleic acids, stained on a Fluidics Station 450 (Affymetrix), and scanned on a GeneChip Scanner 3000 7G system (Affymetrix).

Thirty-six samples (three replicates per treatment including control plants) were analysed at the Genomics Unit of the Spanish National Centre for Biotechnology (CNB-CSIC, Madrid). RNA integrity analyses were done with an Agilent’s Bioanalyzer 2100 using the NanoChip protocol [8 (link)]. The custom GrapeGen Affymetrix GeneChip® (A-AFFY-162 and GPL11004 ArrayExpress and GEO accession numbers, respectively) [24 (link)], was processed as previously described [74 (link)]. Briefly, biotinylated RNA was prepared from 2 μg of total RNA according to the standard Affymetrix protocol. After the first and second strand synthesis of cDNA in vitro transcription was performed using T7 RNA polymerase and biotinylated nucleotides, to produce biotin-labeled cRNA. Labelled cRNA was fragmented to the 50–200-bp size range, and each sample was added to a hybridization solution containing 100 mM 2-(N-morpholino) ethanesulfonic acid, 1 M Na+, and 20 mM of EDTA in the presence of 0.01 % of Tween-20 to a final cRNA concentration of 0.05 μg/ml. Hybridization was performed for 16 h at 45 °C [8 (link)]. Each GeneChip was washed and stained with streptavidin-phycoerythrin in a Fluidics Station 450 (Affymetrix) following the EukGE-WS2v5 script. After washing, the chips were scanned at 1.56 μm resolution in a GeneChip® Scanner 3000 7G System (Affymetrix). The software used was GeneChip Operating Software.

Total RNA was isolated using the RNeasy Micro Kit (Qiagen, Venlo, The Netherlands);
cDNA was synthesized using MMLV reverse transcriptase and random primers (Life
Technologies) according to manufacturer's instructions. Real-time PCR was carried out
using a StepOnePlus real-time PCR system (Applied Biosystems, Foster City, CA) with
SYBR Green PCR Master Mix (Applied Biosystems) and the appropriate primer pairs
(Supplementary file
6; Integrated DNA Technologies). Relative mRNA abundance of target genes
was determined by subtracting the threshold cycle for the internal reference
(Hprt1) from that of the target. Primer pairs were tested for
linear amplification over two orders of magnitude.
For microarray analysis, cDNA was prepared using the Ovation Pico WTA System V2 and
labeled using the Encore Biotin Module (NuGEN, San Carlos, CA). Labeled cDNA
libraries were hybridized to Affymetrix Mouse 1.0 ST arrays (Affymetrix, Santa Clara,
CA) and scanned with the Affymetrix GeneChip Scanner 3000 7G System. Raw data were
normalized by RMA and probesets mapped to unique Entrez Gene IDs using a custom
Brainarray CDF. The limma R package was used for analyzing differential gene
expression. GENE-E software (Broad Institute) was used for heat map generation and
hierarchical clustering.

Yeast cells were grown in YPD medium to mid-log phase and were collected by centrifugation. Total RNA was extracted from whole cell lysates using hot phenol and RNA (200 ng) was used for first strand cDNA synthesis using the GeneChip^® 3′ IVT Express Kit (Affymetrix, Santa Clara, CA, USA). aRNA (7.5 μg) was hybridized for 16 hr at 45 °C in a GeneChip Yeast Genome 2.0 Array. GeneChips were washed and stained in a Affymetrix Fluidics Station 450, and then scanned using the GeneChip Scanner 3000 7G System. The data were analyzed using Microarray Suite version 5.0 (MAS 5.0) with Affymetrix default analysis settings.
The accession number of the microarray data is GSE220290.