Low rna input linear amplification kit

RNA was hybridized to an Agilent Whole Mouse Gene Expression Microarray (G4852A, 8 × 60K; Agilent Technologies), and one-color gene expression was performed according to the manufacturer’s protocol. Labeled cRNA was synthesized from 100 ng total RNA using the Low RNA Input Linear Amplification kit (Agilent Technologies) in the presence of cyanine 3-cytosine triphosphate (CTP; PerkinElmer, Boston, MA, USA). Images generated by the Agilent scanner and Feature Extraction 10.5 software (Agilent Technologies) were used to obtain the microarray raw data. Qlucore Omics Explorer software (QOE) (http://www.qlucore.com/; Qlucore, Lund, Sweden) was used to analyze the microarray data.

Agilent whole genome human oligonucleotide microarrays which contain 44 thousand 60-mer oligonucleotides representing over 41 thousand probes and transcripts was used for whole transcriptome analysis. The Human Universal Reference RNA from Stratagene (Santa Clara, CA, USA) served as a control to standardize hybridization levels between microarrays and was coupled to Cy5. Two hundred nanograms of total RNA from the KCOT samples were converted into labeled cRNA with nucleotides coupled to fluorescent dye Cy3 using the Low RNA Input Linear Amplification Kit (Agilent Technologies, Palo Alto, CA). The Cy3-labeled cRNA (1.65 ng) from each sample and Cy5 coupled universal reference RNA was hybridized to whole genome array formatted chips. The hybridized array was washed, scanned and data extracted from the scanned image using Feature Extraction version 9.5 (Agilent Technologies, Palo Alto, CA).
The microarray data were submitted to the Gene Expression Omnibus (GEO) microarray database (accession number GSE68532).

Complementary RNA was generated from 100 ng total RNA (in the case of serum, 100 ng total RNA was obtained by mixing three samples) and labeled with cyanine 3 using a Low RNA Input Linear Amplification Kit (Agilent Technologies). Labeled cRNA was hybridized to Whole Rat Genome Oligo Microarrays (4×44K) using a Gene Expression Hybridization Kit (Agilent Technologies). The Whole Rat Genome Oligo Microarray (4×44K) contains 41,090 probes (not including control probes). Signals were detected with an Agilent DNA Microarray Scanner, and the scanned images were analyzed using Feature Extraction Software (ver. 9.5.3.1).

Gene expression was measured using a SurePrint G3 Rat GE8x60K Microarray Kit (Agilent Technologies, CA, USA) containing 62,976 oligonucleotide probes representing 30,003 genes. Total RNAs (200 ng) were labelled with cyanine 3-CTP using a Low RNA Input Linear Amplification Kit according to the manufacturer’s protocol (Agilent Technologies, CA, USA). For each reaction, the cRNA yield and specific activity of cRNA were determined using a NanoDrop ND-1000 spectrophotometer. Only the cRNAs with yields of > 0.9 μg and specific activities > 6.0 pmol of dye per microgram of cRNA were used for hybridisation. The labelled cRNAs were hybridised to microarray slides (eight arrays per slide) following the Agilent One-Color microarray-based gene expression analysis protocol (Agilent Technologies, CA, USA). The slides were scanned (8 × 60 k array slides at 3 μm resolution) using an Agilent DNA microarray scanner (G2505C) and colour setting of an Agilent G3_GX_1. The scanned images were analysed using Feature Extraction Software 10.5 (Agilent).

Gene expression was measured using Agilent Whole Rat Genome Microarrays (4 x 44k format; Agilent Technologies, Inc.). Total RNAs (500ng) were labeled with cyanine dye (Cy3-CTP) using the Low RNA Input Linear Amplification Kit according to the manufacturer’s protocol (Agilent Technologies, Inc.). For each reaction, cRNA yields and specific activities were determined using the NanoDrop ND-1000 Spectrophotometer. Only the cRNAs with yields >1.65 µg and specific activities > 9.0 pmol of dye per µg cRNA were used for hybridization. Labeled cRNAs were hybridized to microarray slides (4 arrays per slide) following the Agilent One-Color Microarray-Based Gene Expression Analysis Protocol (V5.5, Agilent Technologies, Inc.). The hybridized slides were scanned using a GenePix 4000B scanner (Axon Instruments, Sunnyvale, CA) at 5µm resolution using appropriate photomultiplier tube gain settings to obtain the highest intensity with <1% saturated pixels. The resulting images were analyzed by quantifying the Cy3 fluorescence intensity at each of the 45,018 gene spots (features) on each array using the Agilent Feature Extraction Software (V9.5). The median fluorescence intensity of all the pixels within each feature was taken as the intensity value for that feature.