One color rna spike in kit

Cells (1 × 10⁹ cells) were harvested at two time points - 5 g/L and 50 g/L of CO₂ released - by centrifugation at 1000 g for 5 min at 4 °C and the cell pellets were washed with DEPC-treated water and then frozen in methanol at −80 °C. Total RNA was extracted with Trizol reagent (Gibco BRL, Life Technologies) and was purified with the RNeasy kit (Qiagen). The quantity and the quality of the extracted RNA were checked by spectrometry (NanoDrop 1000, Thermo Scientific). We used the Agilent 8x15k gene expression microarrays (Design ID 016322, Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s instructions. Fluorescent cRNAs were synthesized from 100 ng of total RNA using the One color RNA Spike-In kit (Agilent Technologies). Labeled cRNA was purified with the RNeasy kit (Qiagen). Microarrays were hybridized for 17 h at 65 °C in a rotating hybridization oven (Corning), with the Gene Expression Hybridization kit (Agilent). The hybridization signal was detected with a GenePix 4000B laser scanner (Axon Instruments).

Five milliliters of peripheral venous blood was collected from
each subject. RNA was isolated using QIAamp® RNA Blood
Mini Kit (Qiagen) and its quality was assessed using Agilent 2100
Bio-analyzer, version G2938C. cDNA synthesis, cRNA synthesis
and cyanine 3 (Cy3) labeling was done using Agilent one color
RNA spike-In kit and Agilent Quick Amp Kit, One-Color.
Quality checked Cy3 labeled cRNA samples were hybridized to
microarray slides (Agilent platform GPL13497), followed by
incubation of slides at 65°C for 17 hours and washing. Microarray
data was captured using Agilent Feature Extraction Software
(Version 9.5.3). Data pre-processing and mining were performed
using GeneSpring GX (Version 12.6.1). Gene expression was
compared among three subject groups using unpaired t-test and
one-way ANOVA. Statistical analysis was done to find out
differentially expressed genes (corrected p-value of &0.05) with
minimum two-fold change in expression. Microarray based gene
expression data was uploaded on Gene Expression Omnibus
(http://www.ncbi.nlm.nih.gov/geo/) with accession number
GSE72999.

The miRNA profiling data was extracted from our group’s previous publication^{33 (link)}. For gene expression profiling was done using the SurePrint G3 mouse Gene Expression v1 8 × 60 K arrays, the Low Input Quick Amp Labeling One-Color Kit (Agilent Technologies, USA) and the One-color RNA Spike-in Kit (Agilent Technologies) following the manufacturer’s standard protocol. 100 ng of total RNA input was used. After cDNA synthesis, amplification, labeling and purification, the reaction efficacy was checked using the NanoDrop (Thermo Scientific, USA). The hybridization was performed for 17 hours at 10 rpm and 65 °C in a Hybridization Oven (Robbins Scientific, USA). Slide wash was done according to the manufacturer’s recommendation. Cy3 intensities were detected by one-color scanning using Agilent DNA microarray scanner at 3 micron resolution. Raw data from scanned image files were extracted using Feature Extraction software (10.7.3.1). Spike-in used in the experiment gave reliable data and all arrays passed the quality control.