Total RNA from the proteinase K-treated microdissectate was extracted using FFPE RNA Isolation Kit from Norgen Biotek® (Thorold, Canada). 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. Two-hundred-fifty or 400 ng of RNA, as quantified using RiboGreen assay, 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.
Total RNA from the proteinase K-treated microdissectate was extracted using FFPE RNA Isolation Kit from Norgen Biotek® (Thorold, Canada). 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. Two-hundred-fifty or 400 ng of RNA, as quantified using RiboGreen assay, 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 6th generation miRCURY™ LNA microRNA arrays (Exiqon®, Vedbaek,Denmark) that were used have approx. 2383 locked nucleic acid oligonucleotide probes on quadruplicate 100 µM-sized probe-spots to detect all 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. '1_Exiqon*.txt' and '0_Exiqon*.txt' represent the raw data for Hy3 and Hy5, respectively.
Data processing
Data processing was done in R (version 2.12.1) using the limma (version 3.6.9) 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. For probe-set summarization, done separately for the Hy3™ and Hy5™ signal values, 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.
