Total RNA was isolated from blood collected and stored in PAXgene Blood RNA tubes using a PAXgene Blood RNA isolation kit at the University of Pennsylvania as per the protocol suggested by the manufacturer (Qiagen®, Valencia, CA, USA). The human universal reference RNA was made by pooling contents of FirstChoice total RNA panel that is made of RNA from different tissues of multiple individuals (Ambion®, Austin, TX, USA; catalog no. AM6000.
Label
Hy3
Label protocol
RNA was labeled by Exiqon®, Vedbaek, Denmark using the miRCURY LNA microRNA Power labeling kit. Five hundred ng of RNA, as quantified using Bioanalyzer (Agilent®, Santa Clara, CA, USA), was used per labeling reaction with the Hy3 dye. A human universal reference RNA, made by pooling contents of FirstChoice total RNA panel (Ambion®, Austin, TX, USA; catalog no. AM6000), was similarly labeled with the Hy5 dye. Synthetic small RNAs were spiked-in to the RNA samples before labeling as per the kit protocol.
sample type: universal reference RNA was made by pooling contents of FirstChoice total RNA panel that is made of RNA from different tissues of multiple individuals, Ambion, Austin, TX, USA; catalog no. AM6000 gender: Mixed
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from blood collected and stored in PAXgene Blood RNA tubes using a PAXgene Blood RNA isolation kit at the University of Pennsylvania as per the protocol suggested by the manufacturer (Qiagen®, Valencia, CA, USA). The human universal reference RNA was made by pooling contents of FirstChoice total RNA panel that is made of RNA from different tissues of multiple individuals (Ambion®, Austin, TX, USA; catalog no. AM6000.
Label
Hy5
Label protocol
RNA was labeled by Exiqon®, Vedbaek, Denmark using the miRCURY LNA microRNA Power labeling kit. Five hundred ng of RNA, as quantified using Bioanalyzer (Agilent®, Santa Clara, CA, USA), was used per labeling reaction with the Hy3 dye. A human universal reference RNA, made by pooling contents of FirstChoice total RNA panel (Ambion®, Austin, TX, USA; catalog no. AM6000), was similarly labeled with the Hy5 dye. Synthetic small RNAs were spiked-in to the RNA samples before labeling as per the kit protocol.
Hybridization protocol
Hybridization of the mix of Hy3-labeled sample and Hy5-labeled reference RNAs to the arrays at 56 ºC for 16 hours followed by washing was performed by Exiqon® (Vedbaek, Denmark) on an HS Pro instrument (Tecan®, Männedorf, Switzerland). The 5th generation miRCURY LNA microRNA arrays (Exiqon®, Vedbaek,Denmark) that were used have approx. 1891 locked nucleic acid oligonucleotide probes on quadruplicate 105 µM-sized probe-spots to detect 74%, 72% and 68% of the 1223, 1055 and 680 human, mouse and rat microRNAs, respectively, of miRbase v.16.
Scan protocol
Arrays were scanned, after hybridization and washing, using the G2505B scanning system (Agilent®, Santa Clara, CA, USA) by Exiqon® (Vedbaek, Denmark).
Description
Hy3 and Hy5 are Cy3- and Cy5-like dyes, respectively.
Data processing
Data processing was done in R (version 2.12.1) using the limma (version 3.4.5) Bioconductor package. The 'normexp' method with offset=10 was used for background-correction, and followed with within-array global 'loess' normalization, with span=1/3 and values from probe-spots with flag-values >1 ignored, and then, between-array 'Rquantile' normalization. For probe-set summarization, the median of the values from multiple spots was used when the ratio of the maximum and minimum values was > 1.5; else, the mean was used. The ratios of the summarized normalized Hy3/Hy5 (test/reference) signal-intensities were log2-transformed.
