Agilent scanner g2505c

For microarray analysis, total RNA from serum was amplified and transcribed into fluorescent complementary RNA (cRNA) using the manufacturer's Agilent's Quick Amp Labeling protocol (version 5.7, Agilent Technologies, Santa Clara, CA, USA). The cRNA were hybridized onto the LncPath™ Human Cancer Array (8*15K, Arraystar, Rockville, MD, USA). After washing the slides, the arrays were scanned with the G2505C Agilent Scanner. Images were analyzed by Agilent Feature Extraction software. Quantile normalization and data processing were performed in R. The microarray work was performed by Arraystar.

Total RNA was extracted from mouse aortic tissue using TRIzol LS Reagent (Invitrogen life technologies), RNA concentration and purity were determined by NanoDrop® ND-1000, and the RNase R-treated samples were subjected to labeling reactions and array hybridization [11 (link)]. The data were scanned with an Agilent Scanner G2505 C (Agilent, Santa Clara, CA, USA) using an Arraystar version 2.0 mouse circRNA microarray, the raw data were read by Agilent Feature Extraction software (v11.0.1.1) (Agilent, Santa Clara, CA, USA.), and the R software limma package normalizes the qualified data of quality control and analyzes the standardized gene chip data to obtain differential circRNAs expression profile. CircRNAs were identified to be differentially expressed (fold change >1.5; P < 0.05) [12 (link)].

Total RNA was extracted from PRMT1-NC1, PRMT1-NC2, PRMT1-KD1, PRMT1-KD2, SMARCA4-KD1, and SMARCA4-KD2 HCT116 cell lines. Total RNA was quantified by the NanoDrop ND-2000 (Thermo Scientific), and RNA integrity was assessed using an Agilent Bioanalyzer 2100 (Agilent Technologies). Sample labeling, microarray hybridization, and washing were performed based on the manufacturer’s standard protocols. Briefly, total RNA were transcribed to double strand cDNA, then synthesized into cRNA and labeled with Cyanine-3-CTP. The labeled cRNAs were hybridized onto the microarray. After washing, the arrays were scanned by the Agilent Scanner G2505C (Agilent Technologies). The 60-mer oligo nucleotide probes were designed using a microarray (Agilent) and performed by Oe-biotech (Shanghai, China). For the study of differential gene expression, Genespring (version13.1, Agilent Technologies) were employed to complete the basic analysis with the raw data. The genes with a fold change value greater than 1.5, and a p value < 0.01 was considered differentially expressed. Relationships of differentially expressed genes were determined by GO and GSEA analysis.

Microglial exosomes acquired from the injured brain were sent for miRNA microarray analysis (OE Biotechnology, Shanghai, China). Total RNA was quantified by the NanoDrop ND-2000 (Thermo Fisher Scientific), and the RNA integrity was assessed using the Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). The sample labeling, microarray hybridization, and washing were performed in accordance with the manufacturer’s standard protocols. Briefly, total RNA was dephosphorylated, denaturated, and labeled with cyanine-3-CTP. After purification, the labeled RNAs were hybridized onto the microarray that contains 1,902 probes for mature miRNA. Then, the arrays were scanned with the Agilent Scanner G2505C (Agilent Technologies).
We selected 3, 14, and 42 DPI to represent, respectively, the time points of acute phase, sub-acute phase, and chronic phase after rmTBI. The miRNAs with level change of more than 2-fold (up- or downregulated compared with the Sham group) at the 3 time points (p < 0.01) were screened out, and qRT-PCR was performed to validate the expression changes of miR-124-3p in injured brain after rmTBI.

Pre-amplification of cDNA was performed with miRNA specific primers as provided with the Megaplex™ Primer Pools, Human Pools Set v3.0 (Applied Biosystems). Fluorescently-labeled miRNA were prepared according to Agilent protocol “miRNA Complete Labeling and Hyb Kit”. 100 ng labeled miRNA sample were hybridized for at least 20 h at 55 °C on Agilent human miRNA Microarray Release 21.0, 8x60k. The cDNAs were then diluted and loaded onto a TaqMan® Array Human MicroRNA A + B Cards Set (Applied Biosystems) and qRT-PCR run performed. RNU6-2_11 and RNU6_11 served as housekeeping genes on all Taqman Low Density Array (TLDA) cards run. Fold changes in expression were calculated for all miRNAs by dividing mean values from group B with group A. Gene Expression Microarrays were scanned using the Agilent Scanner G2505C. The scanned images were analyzed with Feature Extraction Software (Agilent technologies) using default settings.

“IP” RNAs and “Sup” RNAs were added with an equal amount of calibration spike-in control RNA, separately amplified, and labeled with Cy3 (for “Sup”) and Cy5 (for “IP”) using the Arraystar Super RNA Labeling Kit (Rockville, MD, USA). The synthesized cRNAs were purified by the RNeasy Mini Kit (Hilden, German). The concentration and specific activity (pmol dye/μg cRNA) were measured with NanoDrop ND-1000 (Thermo Fisher Scientific, Waltham, MA, USA). 2.5 μg of Cy3 and Cy5 labeled cRNAs were mixed. The cRNA mixture was fragmented by adding 5 μL of Blocking Agent and 1 μL of 25× Fragmentation Buffer, heated at 60 °C for 30 min, and combined with 25 μL of 2× Hybridization buffer. 50 μL of hybridization solution was dispensed into the gasket slide and assembled to the m6A-mRNA&lncRNA Epitranscriptomic Microarray slide. The slides were incubated at 65 °C for 17 h in an Agilent Hybridization Oven (Agilent Technologies, Santa Clara, CA, USA). The hybridized arrays were washed, fixed, and scanned using an Agilent Scanner G2505C (Agilent Technologies, Santa Clara, CA, USA).