Genechip scanner 3000 7g

The expression profile of dopamine-related genes was assessed using HG-U133A 2.0 microarrays (Affymetrix, Santa Clara, CA, USA), the GeneChip™ 3′IVT PLUS, and Ge-neChip™ HT 3′IVT PLUS Reagent kits (ThermoFisher Scientific, Waltham, MA USA, Cat No. 902416, 902417). Fluorescence intensity was measured with the Gene Array scanner (Agilent Technologies, Santa Clara, CA, USA). The phrase “dopamine” was entered in the Affymetrix NetAffx™ Analysis Center database (http://www.affymetrix.com/analysis/index.affx; accessed on 1 August 2021) to obtain probe names and identification numbers.
The expression profile of miRNAs in endometrial tissue samples was determined using GeneChip miRNA 2.0 microarrays (Affymetrix, Santa Clara, CA, USA), according to the manufacturer’s protocol. The GeneChip Scanner 3000 7G (Agilent Technologies, Santa Clara, CA, USA) was used to scan the microarrays. A TargetScan prediction tool (http://www.targetscan.org, accessed on 1 August 2021) was then used to determine which miRNAs differentiating endometrial cancer from the control could potentially affect the expression of dopamine-related mRNAs.

Total RNA was extracted from exosomes using TRI reagent (Molecular Research Center, Inc., Cincinnati, OH, USA) following the manufacturer’s guidelines. Labelling was performed using 250 ng of total RNA. Each RNA strand was reacted with RNA polymerase for poly-A tailing and ligation was performed with a biotin-labelled 3DNA dendrimer. Reacted RNA strands were hybridized to an Affymetrix GeneChip miRNA 4.0 Array (Affymetrix, Santa Clara, CA, USA) for 18 hours at 48 °C. The chip was washed and stained using an Affymetrix Fluidics Station 450. After amplification, fluorescence signals were detected with an Affymetrix GeneChip Scanner 3000 7 G. The results were analysed with an Agilent scanner and associated software. The expression levels of miRNAs were analysed using Expression Console 1.4.1 (Affymetrix). For normalization of miRNA expression, we performed a quantile normalization using GeneSpring GX 13.1.1 (Agilent technologies, Santa Clara, CA, USA).

MiRNAs were extracted using the mirVana miRNA isolation kit (Ambion, Austin, TX, USA) according to the manufacturer's protocols. Purified miRNAs were labelled using the mirVana miRNA Array Labeling kit and coupled to the Cy5 Post-Labeling Reactive Dye (Amersham, GE Healthcare Bio-Sciences, Piscataway, NJ, USA). The labelled samples were washed and hybridized in duplicate to mirVana miRNA Bioarrays (Ambion) using the mirVana miRNA Bioarray Essentials kit. Fluorescence intensities were processed and measured using the GeneChip scanner 3000 7G (Agilent Technologies, Santa Clara, CA, USA). The levels of miRNA hybridization were determined using GenePix Pro 6.0 software as recommended by the manufacturer. The background-adjusted intensity for each miRNA was subjected to a global variance stabilization normalization procedure. MiRNAs were considered to be overexpressed only if the differences were determined to be significant by a two-sample t-test (P < 0.05) and on average showed at least a 1.5-fold increase in patient samples compared with matched controls. Heatmap analysis and hierarchical clustering were performed with R project software.