Low rna input fluorescent linear amplification kit

Microarray analysis were performed using the Whole Human Genome (8 × 60 k, Design ID 39494) Oligo Microarray according to the Agilent 60-mer Oligo Microarray Processing Protocol (Agilent Technologies). Total RNA samples (200 ng) were used to prepare Cy3-labeled cRNA using a Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies). Fluorescence-labeled cRNAs were purified using an RNeasy RNA Purification Kit (Qiagen Inc., Hilden, Germany). Two independent RNA samples were used to confirm the reproducibility of the microarray analyses. The images were analyzed using the Feature Extraction Software (Ver. 10.7.3.1) and GeneSpring GX 12.1 software (Agilent Technologies). Normalization was performed as follows: (1) intensity-dependent Lowess normalization; (2) data transformation, with measurements set to ≤ 0.01; (3) per-chip 75th-percentile normalization of each array; and, (4) per-gene: normalized to the median of each gene. Genes differently expressed more than twice between Non-sh and shCBX6–8 treated Meso-4 cells were selected. The raw and processed data were deposited in the Gene Expression Omnibus database (access ID: GSE126605).

Each yeast strain had two independent fermentations and three samples were taken from each independent fermentation. Cells from each yeast strain fermentation were pelleted by centrifugation (4000 rpm/min, 5 min), at 12°C and 28°C. Cells were collected at the beginning of the exponential phase by taking samples two generations after inoculation. The RNA extraction protocol was based on subsequent treatments with phenol-tris, phenol-chloroform (5:1) and chloroform-isoamyl alcohol (24:1), and an ethanol precipitation with sodium acetate
^{13 (link)}. RNA concentrations and purity were determined using a Nanodrop spectrophotometer ND-1000 (Nanodrop Technologies™, Wilmington, DE). RNA integrity was checked by electrophoresis in agarose gel (1%). 2–4 μg of total RNA from each sample was linearly amplified using the Low RNA Input Fluorescent Linear Amplification kit (Agilent Technologies™, Ca, USA). 2–3 µg of amplified cRNA was used as template for cDNA synthesis. cDNA was marked indirectly using the SuperScript™ Indirect cDNA Labeling System (Invitrogen™, San Diego, CA). Cy3 and Cy5 mono-reactive Dye (Amersham GE Healthcare™, Amersham UK) were used as the fluorophores and dye incorporation was monitored using a Nanodrop spectrophotometer.

Cells were collected by centrifugation (4000 rpm/min, 5 min) from two independent fermentations done at 12°C and 28°C at the beginning of stationary phase, and determined when 50% of the reducing sugars were consumed. The RNA extraction method was based on consecutive treatments with phenol-tris, phenol-chloroform (5∶1) and chloroform-isoamyl alcohol (24∶1), and a final precipitation with ethanol and sodium acetate [39] . RNA concentrations and purity were determined using a Nanodrop spectrophotometer ND-1000 (Nanodrop Technologies, Wilmington, DE, USA). RNA integrity was determined by electrophoresis in 1% agarose gel. 2–4 µg of total RNA from each sample was linearly amplified using the Low RNA Input Fluorescent Linear Amplification kit (Agilent Technologies, CA, USA). Then 2–3 ug of amplified cRNA were used as a template for cDNA synthesis. cDNA was marked indirectly with the “SuperScript Indirect cDNA Labeling System” (Invitrogen, San Diego, CA, USA). The fluorophores used were Cy3 and Cy5 mono-reactive Dye (Amersham GE Healthcare, Amersham, UK) and dye incorporation was monitored by a Nanodrop spectrophotometer.

RNA from three biological replicates of control or UV irradiated Ewing Sarcoma cells (either SK-N-MC or LAP-35) and HEP3B cells collected 6 hours after the treatment was purified using RNeasy Mini kit (Qiagen) and digested with DNase RNase free (Qiagen). cDNA and Cy5-Cy3 labelled cRNA were generated from the total RNA using the Agilent Low RNA Input Fluorescent Linear Amplification kit. 8 μg of each cRNA were used for the hybridization with the arrays (Agilent In situ hybridization kit plus). After hybridization, arrays were washed, and scanned images analyzed as previously described [14 (link)]. Three biological replicates were hybridized, with both direct and dye-reversal hybridizations. General gene expression values represent the average of log2 ratios for all the probes of a locus. Statistical analyses were carried out with Linear Models for Microarray Data (Limma; Bioconductor Project) [51 (link)]. The background correction method used in the analysis was Normexp [52 (link)]. Locally weighted linear regression (LOWESS) analysis was used as a normalization method [53 (link)].
The cutoff considered was fold change >|1,3| for gene expression changes and fold change >|1,4|; (Z-SCORE >3; p-value <0,01) for alternative splicing changes. The data were deposited in GEO database with the accession number GSE59889.