tissue: squamous cell carcinoma stroma patient: 28 recurrence: N histology: SCC age_at_surgery: 64 gender: M race: White smoking_history: former pathologic_stage: 1A months_to_death_or_censorship: 51 tumor_component: S
Treatment protocol
Laser capture microdissection was performed to isolate epithelial or stromal compartments of tumors using hematoxylin-eosin-stained tissue sections. Refer to Patnaik et al., BMC Research Notes, 2012, 5:40 (PMID 22260539) for details on tissue section preparation and microdissection.
Growth protocol
Formalin-fixed and paraffin-embedded, resected, stage I, human lung cancer tumor tissue samples were provided by the Department of Pathology of Roswell Park Cancer Institute, Buffalo, NY, USA.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from microdissectates using the FFPE RNA Purification kit (catalog# 25300, Norgen-Biotek®, Thorold, ON, Canada) following the kit manufacturer's guidelines. The kit uses silicon carbide spin-columns. The extracted RNA preparations were not treated with DNase. The low-range Quant-iT™ Ribogreen assay (Life Technologies®, Carlsbad, CA) was used to estimate RNA concentration using yeast tRNA as the standard. Refer to Patnaik et al., BMC Research Notes, 2012, 5:40 (PMID 22260539) for details on RNA isolation and quantitation. The reference RNA, a human 'universal reference total RNA,' was obtained by Exiqon® from Ambion® (catalog# AM6000; Austin, TX).
Label
Hy3
Label protocol
The miRCURY™ LNA microRNA Hi-Power Labeling kit from Exiqon® was used to label 350 ng of test RNA with the Cy3-like Hy3™ dye. The reference RNA (350 ng) was similarly labeled with the Cy5-like Hy5™ dye. Artificial small RNAs were spiked in to test and reference RNA samples before labeling. This work was performed as a commercial service by Exiqon®.
Laser capture microdissection was performed to isolate epithelial or stromal compartments of tumors using hematoxylin-eosin-stained tissue sections. Refer to Patnaik et al., BMC Research Notes, 2012, 5:40 (PMID 22260539) for details on tissue section preparation and microdissection.
Growth protocol
Formalin-fixed and paraffin-embedded, resected, stage I, human lung cancer tumor tissue samples were provided by the Department of Pathology of Roswell Park Cancer Institute, Buffalo, NY, USA.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from microdissectates using the FFPE RNA Purification kit (catalog# 25300, Norgen-Biotek®, Thorold, ON, Canada) following the kit manufacturer's guidelines. The kit uses silicon carbide spin-columns. The extracted RNA preparations were not treated with DNase. The low-range Quant-iT™ Ribogreen assay (Life Technologies®, Carlsbad, CA) was used to estimate RNA concentration using yeast tRNA as the standard. Refer to Patnaik et al., BMC Research Notes, 2012, 5:40 (PMID 22260539) for details on RNA isolation and quantitation. The reference RNA, a human 'universal reference total RNA,' was obtained by Exiqon® from Ambion® (catalog# AM6000; Austin, TX).
Label
Hy5
Label protocol
The miRCURY™ LNA microRNA Hi-Power Labeling kit from Exiqon® was used to label 350 ng of test RNA with the Cy3-like Hy3™ dye. The reference RNA (350 ng) was similarly labeled with the Cy5-like Hy5™ dye. Artificial small RNAs were spiked in to test and reference RNA samples before labeling. This work was performed as a commercial service by Exiqon®.
Hybridization protocol
Hybridizations and washes were performed according to the Exiqon® miRCURY™ LNA microRNA Array Instruction manual using an HS4800 hybridization station (Tecan®, Grödig, Austria). This work was performed as a commercial service by Exiqon®.
Scan protocol
Arrays were scanned using the G2565BA Microarray Scanner System (Agilent®), and image analysis was carried out using the ImaGene™ software (version 9; BioDiscovery®, Hawthorne, CA). This work was performed as a commercial service by Exiqon®.
Description
Sample 60.
Data processing
Raw Hy3™ and Hy5™ signal data for all 136 arrays were processed together in R (version 2.15.1) using the limma Bioconductor package (version 3.12.3) on Mac OS X 10.6.8. Briefly, background signals were adjusted for using the 'normexp' method with 'offset' value of 10. Array data was then subjected to within-array global loess normalization with a 'span' value of 1/3. This was followed by between-array 'quantile' normalization. Probe-set summarization for Hy3™/Hy5™ ratio values (test RNA/reference RNA) was then performed, using the mean value from replicate probe-spots when the maximum was <1.5x minimum value, using the median value otherwise.
