age: 48 gender: Male cell type: Hepatocellular carcinoma cancer stage: 2 overall survival (month): 47 disease free survival (month): 47
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from the frozen tissues using Trizol (Life Technologies, Inc., Carlsbad, CA, USA) according to the manufacturer’s protocol.
Label
Cy5
Label protocol
Double-strand cDNA (ds-cDNA) was synthesized from 5 μg of total RNA using SuperScript ds-cDNA synthesis kit (Life Technologies, Inc., Carlsbad, CA, USA) in the presence of 100 pmol oligo dT primers. ds-cDNA was cleaned and labeled in accordance with the NimbleGen Gene Expression Analysis protocol (NimbleGen Systems, Inc., USA). Briefly, ds-cDNA was incubated with 4 μg RNase A at 37°C for 10 min and cleaned using phenol:chloroform:isoamyl alcohol, followed by ice-cold absolute ethanol precipitation. The purified cDNA was quantified using a NanoDrop ND-1000. For Cy3/Cy5 labeling of cDNA, the NimbleGen Dual-Color DNA labeling kit was used according to the manufacturer's guideline detailed in the Gene Expression Analysis protocol (NimbleGen Systems, Inc., Madison, WI, USA).
sample type: Reference RNA pool from 20 normal livers
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from the frozen tissues using Trizol (Life Technologies, Inc., Carlsbad, CA, USA) according to the manufacturer’s protocol.
Label
Cy3
Label protocol
Double-strand cDNA (ds-cDNA) was synthesized from 5 μg of total RNA using SuperScript ds-cDNA synthesis kit (Life Technologies, Inc., Carlsbad, CA, USA) in the presence of 100 pmol oligo dT primers. ds-cDNA was cleaned and labeled in accordance with the NimbleGen Gene Expression Analysis protocol (NimbleGen Systems, Inc., USA). Briefly, ds-cDNA was incubated with 4 μg RNase A at 37°C for 10 min and cleaned using phenol:chloroform:isoamyl alcohol, followed by ice-cold absolute ethanol precipitation. The purified cDNA was quantified using a NanoDrop ND-1000. For Cy3/Cy5 labeling of cDNA, the NimbleGen Dual-Color DNA labeling kit was used according to the manufacturer's guideline detailed in the Gene Expression Analysis protocol (NimbleGen Systems, Inc., Madison, WI, USA).
Hybridization protocol
Microarrays were hybridized at 42°C during 16 to 20h with 4 μg of Cy3/Cy5 labeled ds-cDNA in NimbleGen hybridization buffer/hybridization component A in a hybridization chamber (Hybridization System - NimbleGen Systems, Inc., Madison, WI, USA). Following hybridization, washing was performed using the NimbleGen Wash Buffer kit (NimbleGen Systems, Inc., Madison, WI, USA).
Scan protocol
Microarrays were scanned at 5 µm resolution using a GenePix 4000B microarray scanner piloted by GenePix Pro 6.0 software (Axon Instruments, Union City, CA). Scanned images were then imported into NimbleScan software (version 2.5) for grid alignment and raw intensity extraction.
Description
The HCC patients had not received adjuvant chemotherapy.
Data processing
The raw microarray data sets were normalized in Agilent GeneSpring GX software (version 11.5) (LOWESS normalization). After normalization, the probes with signal greater than 200 in more than 18 channels among all 118 Cy5/Cy3 channels were chosen for further analysis. Multiple probes representing the same lncRNA or mRNA were merged by getting a median value. The lncRNAs or mRNAs which had absolute value of log2Ratio>=1.59 in at least 4 arrays across the sample sets were selected for further analyses (8,255 lncRNAs and 12,139 mRNAs).
