cell line: NCI-H460 cell type: human lung large cell carcinoma cells treatment: nicotine timepoint: 24 hours
Treatment protocol
Nictoine (5 uM; Sigma®, Saint Louis, MO) or cigarette smoke extract (5 uM nicotine-equivalent) was added to the culture media, and after 2 hours, cells were irradiated with one dose of 6 Gy ionizing radiation (cesium-137 source), or cisplatin (1 uM; Sigma®) was added to the culture medium. All treatments were performed concurrently using a total of nine 6-well cuture plates. After another 2 or 24 hours, cells were harvested by scraping into the culture medium, spun down, washed in 10 ml phosphate-buffered saline, and collected as cell-pellets after another round of centrifugation at room temperature. The total 54 cell-pellets were stored frozen at -80 ºC until RNA extraction.
Growth protocol
3x10e5 NCI-H460 cells, at passage number 9 after procurement from ATCC® (Manassas, VA) and maintained in RPMI-1640 medium (Life Technologies®, Grand Island, NY) with 10% fetal bovine serum (Atlanta Biologicals®, Lawrenceville, GA) at 37 ºC under 5% carbon dioxide, were seeded per well of 6-well tissue culture dishes. After 24 hours, the culture medium was replaced with that without serum and cells were grown for another 24 hours.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from -80 ºC-frozen cell-pellets using the Total RNA Purification kit (catalog# 37500, Norgen-Biotek®, Thorold, ON, Canada) following the kit manufacturer's guidelines. The kit uses silicon carbide spin-columns. On-column DNase I treatment was performed to reduce contaminating genomic DNA. RNA eluted from the columns was treated on-column with DNase I again as per the manufacturer's guidelines provided with the RNase-free DNase I kit (Norgen-Biotek®). Absorbance at 260 nm and electrophoresis on Bioanalyzer™ 2100 (Agilent®, Austin, TX) was used to estimate RNA concentration and quality. The reference RNA was created by pooling all of the 54 unique RNA samples (at equal concentration).
Label
Hy3
Label protocol
The miRCURY™ LNA microRNA Hi-Power Labeling kit from Exiqon® was used to label 750 ng of test RNA with the Cy3-like Hy3™ dye. The reference RNA (750 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®.
sample type: reference cell line: NCI-H460 cell type: human lung large cell carcinoma cells
Treatment protocol
Nictoine (5 uM; Sigma®, Saint Louis, MO) or cigarette smoke extract (5 uM nicotine-equivalent) was added to the culture media, and after 2 hours, cells were irradiated with one dose of 6 Gy ionizing radiation (cesium-137 source), or cisplatin (1 uM; Sigma®) was added to the culture medium. All treatments were performed concurrently using a total of nine 6-well cuture plates. After another 2 or 24 hours, cells were harvested by scraping into the culture medium, spun down, washed in 10 ml phosphate-buffered saline, and collected as cell-pellets after another round of centrifugation at room temperature. The total 54 cell-pellets were stored frozen at -80 ºC until RNA extraction.
Growth protocol
3x10e5 NCI-H460 cells, at passage number 9 after procurement from ATCC® (Manassas, VA) and maintained in RPMI-1640 medium (Life Technologies®, Grand Island, NY) with 10% fetal bovine serum (Atlanta Biologicals®, Lawrenceville, GA) at 37 ºC under 5% carbon dioxide, were seeded per well of 6-well tissue culture dishes. After 24 hours, the culture medium was replaced with that without serum and cells were grown for another 24 hours.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from -80 ºC-frozen cell-pellets using the Total RNA Purification kit (catalog# 37500, Norgen-Biotek®, Thorold, ON, Canada) following the kit manufacturer's guidelines. The kit uses silicon carbide spin-columns. On-column DNase I treatment was performed to reduce contaminating genomic DNA. RNA eluted from the columns was treated on-column with DNase I again as per the manufacturer's guidelines provided with the RNase-free DNase I kit (Norgen-Biotek®). Absorbance at 260 nm and electrophoresis on Bioanalyzer™ 2100 (Agilent®, Austin, TX) was used to estimate RNA concentration and quality. The reference RNA was created by pooling all of the 54 unique RNA samples (at equal concentration).
Label
Hy5
Label protocol
The miRCURY™ LNA microRNA Hi-Power Labeling kit from Exiqon® was used to label 750 ng of test RNA with the Cy3-like Hy3™ dye. The reference RNA (750 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 32. Biological replicate 2.
Data processing
Raw Hy3™ and Hy5™ signal data for all 58 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 'Rquantile' 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. All arrays appeared to have yielded data of good quality as assessed using criteria suggested by Exiqon®.
Effect of cigarette smoke extract, cisplatin, nicotine and/or ionizing radiation on microRNA expression in the NCI-H460 human lung large cell carcinoma cell line
Effect of cigarette smoke extract, cisplatin, nicotine and/or ionizing radiation on mRNA and microRNA expression in the NCI-H460 human lung large cell carcinoma cell line
Data table header descriptions
ID_REF
VALUE
Log2-transformed Rquantile normalized probeset-summarized Hy3/Hy5 signal ratio (sample/reference fold-change value)
