Genechip scanner 3000 system

Clariom™ D microarray chips (Thermo Fisher Scientific, Oslo, Norway) were used to analyze the salivary EV-RNA samples. The samples were prepared using a GeneChip^TM WT Pico Reagent Kit (Thermo Fisher Scentific, Oslo, Norway, Cat no 902623), in accordance with the manufacturer’s guidelines. The input quantity of total RNA was 3 ng and pre-IVT amplification was performed using 9 PCR cycles. Hela RNA (0.5 ng total RNA) was used as a positive control to verify that the reagents worked as expected, and RNA-free water was used as a negative control to monitor for RNA and DNA contamination. Loaded Clariom^TM D microarrays were incubated in an Affymetrix GeneChip™ 645 hybridization oven for 17 h at 45 °C with a rotation of 60 rpm, then washed and stained in a GeneChip™ Fluidics Station 450 using a GeneChip™ Hybridization, Wash, and Stain Kit (Thermo Fisher Scientific, Oslo, Norway, Cat no 901241). A GeneChip^™ Scanner 3000 System was used to read the generated fluorescence signal values, corresponding to RNA transcript levels, and scanned data were processed with Affymetrix Command Console^® Software version 4.0 (AGCC), producing DAT files ready for bioinformatic processing (all instruments provided by Affymetrix, Santa Clara, CA, USA).

After concentration adjustment and placement into 24-well plates at 1 × 10⁴ cells/well, PMVECs were subjected to the grouped treatments mentioned above and a 24 h routine culture in the 5% CO₂ incubator at 37°C after mixing gently. Firstly, total RNA in PMVECs was sequestered by the use of TRIzol and further was highly purified by virtue of RNeasy kits. Then, a UV spectrophotometer was wielded to measure the RNA level. Secondly, miRCURY™ LNA microRNA Array Power Labeling Kit (Exiqon) was utilized to label the total RNA in cell samples. The results after hybridization were scanned using the GeneChip Scanner 3000 system, and Command Console software v4.0 (Affymetrix) was introduced for reading raw data. Thirdly, after normalization, the differentially expressed miRNAs in the three groups were screened by fold change >2.0 and examined by t-test. The differentially expressed miRNAs screened in each group were intuitively displayed by cluster maps [18 (link)].

RNA samples were labelled using a FlashTag™ Biotin HSR RNA Labeling Kit (Affymetrix). The process begins with a brief tailing reaction followed by ligation of the biotinylated signal molecule to the target RNA sample. Afterward, the biotin-labelled RNA was hybridized onto a GeneChip miRNA 4.0 Array for 42 h at 49 °C using an Affymetrix Hybridization Oven. Using the Affymetrix GeneChip system, GeneTitan Arrays were washed and stained in an Affymetrix Fluidics Station 450 and scanned using an Affymetrix GeneChip Scanner 3000 System. The data were analysed with Expression Console Software using RMA analysis. A filtering step excluding probes not reaching the lower quartile of the coefficient of variation was employed, and the total number of obtained human miRNAs was 4993.