Hybridization oven

We performed RNA labeling and array hybridization according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol with minor modifications as described previously (You et al., 2015 (link)). Firstly, rRNA was removed from the total RNA using mRNA-ONLY™ Eukaryotic mRNA Isolation Kit (Epicentre, Chicago, IL, USA). Then, the purified mRNA was amplified and transcribed into fluorescent cRNA utilizing a random priming method (Arraystar Flash RNA Labeling Kit, Arraystar, Rockville, MD, USA). After purification, the quantity and purity of the labeled cRNAs were measured by NanoDrop ND-1000. 1 μg of each labeled cRNA was fragmented, heated and diluted. 50 μl of hybridization solution was assembled to the lncRNA expression microarray slide and incubated for 17 h at 65°C in an Agilent Hybridization Oven. The hybridized arrays were washed, fixed and scanned using the Agilent DNA Microarray Scanner (part number G2505C).

Before RNA labeling, the Agilent ND-1000 was used to detect RNA degradation and determine RNA concentration. For gene chip analysis, samples were labeled with the Agilent Quick Amp Labeling kit and hybridized with Agilent SureHyb.
Sample labeling and array hybridization were performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technology). Briefly, total RNA from each sample was linearly amplified and labeled with Cy3-UTP. The labeled cRNAs were purified by RNeasy Mini Kit (Qiagen). The concentration and specific activity of the labeled cRNAs (pmol Cy3/μg cRNA) were measured by NanoDrop ND-1000. 1 μg of each labeled cRNA was fragmented by adding 11 μl 10x Blocking Agent and 2.2 μl of 25x fragmentation buffer and then heated at 60°C for 30 min, and finally 55 μL 2x GE hybridization buffer was added to dilute the labeled cRNA. 100 μL of hybridization solution was dispensed into the gasket slide and assembled to the gene expression microarray slide. The slides were incubated for 17 hours at 65°C in an Agilent Hybridization Oven. The hybridized arrays were washed, fixed, and scanned using the Agilent DNA Microarray Scanner (part number G2505C).

Microarrays were performed according to a previously described procedure [30 (link)]. Young buds collected from WT and 5mARF17/WT plants were immediately frozen in liquid nitrogen. A Low-RNA-Input Linear Amplification Kit (Agilent Technologies) was used to amplify and label the total RNA. 5-(3-Aminoallyl)-UTP (Ambion), Cy3 NHS ester (GE Healthcare Biosciences) and Cy5 NHS ester (GE Healthcare Biosciences) were applied following the manufacturers’ instructions. The labeled cRNA was purified using an RNeasy Mini Kit (Qiagen). According to the manufacturer’s instructions, each 44-K Arabidopsis oligo microarray slide was hybridized with 825 ng of Cy3-labeled cRNA and 825 ng of Cy5-labeled cRNA using a gene expression hybridization kit (Agilent) in a hybridization oven (Agilent). The slides were scanned using an Agilent Microarray Scanner (Agilent) and Feature Extraction software 10.7 (Agilent) with the default settings. Three biological replicates of independently grown materials were used. The raw data were normalized with a locally weighted scatter plot smoothing (Lowess) algorithm using Gene Spring Software 11.0 (Agilent).

The expression profiles of exosomal miRNA derived from mixed cytokines and STZ treated or control islets were evaluated using Agilent Mouse miRNA Microarray Based on miRBase Release 21.0 (Agilent technologies, Santa Clara, USA) which analyzes 1881 mature miRNAs totally. Firstly, total 100 ng exosomal RNA was labeled by miRNA Complete Labeling and Hyb Kit (Agilent technologies, Santa Clara, USA). Then, each slide was hybridized with Cy3-labeled miRNA in hybridization Oven (Agilent technologies, Santa Clara, USA) at 55°C, 20 rpm for 20 hours. After hybridization, slides were washed in staining dishes (Thermo Shandon, Waltham, USA) with Gene Expression Wash Buffer Kit (Agilent technologies, Santa Clara, USA) and scanned by Agilent Microarray Scanner (Agilent technologies, Santa Clara, USA). Raw data were normalized by Quantile algorithm included in the R package AgiMicroRna [37 (link)]. After normalization of the original data, the differential expressed miRNAs were screened using fold-change and Student's t-test statistical method. P<0.05 was considered statistically significant.

Total RNA was amplified and labeled using a Low Input Quick Amp Labeling Kit, One-Color (Cat.# 5190-2305, Agilent Technologies). Each slide was hybridized with 1.65 μg of Cy3-labeled cRNA with a Gene Expression Hybridization Kit (Cat.# 5188-5242, Agilent Technologies), maintaining in a Hybridization Oven (Cat.# G2545A, Agilent technologies) for 17 h hybridization. Then, slides were washed with the Gene Expression Wash Buffer Kit (Cat.# 5188-5327, Agilent Technologies) and scanned by an Agilent Microarray Scanner (Cat#G2565CA, Agilent Technologies). Finally, data were extracted with Feature Extraction software 10.7 (Agilent Technologies). Raw data were normalized with the quantile algorithm and limma packages in R.