3d gene human mirna oligo chip

Plasma samples were collected from EDTA-Na blood samples by centrifugation at 1,500×g and stored at -80°C. RNA was extracted from the plasma samples using the 3D-Gene^Ⓡ RNA extraction reagent from a liquid sample kit (TORAY, Tokyo, Japan), according to the manufacturer's instructions. The extracted total RNA was labeled with the 3D-Gene^Ⓡ miRNA labeling kit (TORAY). Labeled RNAs were hybridized onto 3D-Gene^Ⓡ Human miRNA Oligo chips (TORAY) (12 (link)). The annotation and oligonucleotide sequences of the probes conformed to the miRBase miRNA database (http://www.mirbase.org/). After careful washing, the fluorescence signals were scanned with a 3D-Gene^Ⓡ Scanner (TORAY) and were analyzed using the 3D-Gene^Ⓡ Extraction software program (TORAY).
The raw data of each spot were normalized by substitution with the mean intensity of the background signal (determined by the signal intensities of all of the blank spots' and 95% confidence intervals). The measurements of spots with signal intensities that were greater than two standard deviations of the background signal intensity were considered to be valid. The relative expression level of a given miRNA was calculated by comparing the signal intensities of the valid spots throughout the microarray experiments. The normalized data were globally normalized per array, such that the median signal intensity was adjusted to 25.

Polymerase chain reaction (PCR) was performed under the conditions described previously.^{10 –12} (link) The levels of miR-34a, mir-34b, mir-378a, CD44, p53, IL-8, platelet-derived growth factor (PDGF)-ββ, vascular endothelial growth factor (VEGF), and Rho A were normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the results were presented as 2^−ΔCt (relative units of expression). The primers used are listed in Supplementary Table S3 by the assay IDs of the manufacturers.
For miRNA expression profiling, 3D-Gene Human miRNA Oligo Chips (miRBase version 17-21; Toray Industries, Inc., Tokyo, Japan) were used, and the other procedures were the same as those described previously.³³ (link)

RNA extraction was performed as described.7 , 8 (link), 9 (link) For miR expression profiling, 3D-Gene Human miRNA Oligo Chips (miRBase version 17-19; Toray Industries Inc) were used and analyzed as previously described.^{7 ,8} (link) All the data were globally normalized per a microarray, such that the median of the signal intensity was adjusted to 25.

Microarray analysis of the purified RNAs obtained from HuH‐7P‐ and HuH‐7M‐derived exosomes was performed using the Toray microRNA microarray system. Labeled RNAs were hybridized onto 3D‐Gene Human miRNA Oligo chips (v.21; Toray Industries). Raw and processed data from this analysis were deposited in the Gene Expression Omnibus repository (accession number: GSE151169).

Extracted total RNA was labelled with Hy5 using the miRCURY LNA microRNA Array labeling kit (Exiqon, Vedbaek, Denmark). Labelled RNAs were hybridised onto 3D-Gene Human miRNA Oligo chips (Toray, Kamakura, Japan). Fluorescent signals were scanned and analysed using the 3D-Gene Scanner (Toray). The raw data from each spot were normalised by subtraction of the background signal mean intensity, determined by the 95% confidence intervals of the signal intensities of all blank spots. Valid measurements were considered those in which the signal intensity of both duplicate spots was >2 s.d. of the background signal intensity.