tissue: blood gender: male age: 67 y disease state: granuloma of lung lung disease: Non-cancerous lung nodule individual: 94
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from blood collected and stored in PAXgene Blood RNA tubes using a PAXgene Blood miRNA kit at the Roswell Park Cancer Institute, Buffalo, NY, USA 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 Cy3-like 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 Cy5-like Hy5 dye. Sixty-two synthetic small RNAs were spiked-in to the RNA samples before labeling as per the kit protocol.
tissue: Multiple gender: Mixed sample type: Human universal reference RNA from Ambion
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from blood collected and stored in PAXgene Blood RNA tubes using a PAXgene Blood miRNA kit at the Roswell Park Cancer Institute, Buffalo, NY, USA 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 Cy3-like 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 Cy5-like Hy5 dye. Sixty-two 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 C for 16 hours followed by washing was performed by Exiqon (Vedbaek, Denmark) on an HS Pro instrument (Tecan, Männedorf, Switzerland). The seventh generation miRCURY LNA microRNA arrays (Exiqon, Vedbaek, Denmark) that were used have approx. 1892 locked nucleic acid oligonucleotide probes on quadruplicate 100 µM-sized probe-spots (185 µM inter-spot distance) to detect 20 human non-microRNA small RNAs (20 array probes), 25 Exion-proprietary human miRPlus microRNAs (25 probes) and 1916 human miRBase microRNA database microRNAs (1892 probes). Thirty of the 1892+25=1917 probes recognize a total of 72 multiple microRNAs (2-6 per probe). Some of the microRNAs recognized by the 30 are also recognized by other, one-microRNA-specific probes. The array also has probes for viral, mouse and rat microRNAs.
Scan protocol
Arrays were scanned, after hybridization and washing, using the G2505B scanning system (Agilent, Santa Clara, CA, USA) by Exiqon (Vedbaek, Denmark).
Description
files with prefix 1_ are for Hy3 (Cy3) signals and 2_ for Hy5
Data processing
Array data processing was done in R (version 2.14.1) using the limma (version 3.10.2) 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 'quantile' 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.
Whole blood microRNA expression in human lung cancer
Data table header descriptions
ID_REF
VALUE
Log2-transformed ratios of summarized within-array global loess- and between-array quantile-normalized Hy3 and Hy5 signal-intensities. Probe-set summarization was done after calculation of Hy3/Hy5 ratio for each spot.
