|
| Status |
Public on Dec 31, 2014 |
| Title |
Radiation+cigarette_smoke_extract_2h_rep2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
NCI-H460, radiation and cigarette smoke extract, 2 hr
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: NCI-H460
cell type: human lung large cell carcinoma cells
treatment: radiation and cigarette smoke extract
timepoint: 2 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®.
|
| |
|
| Channel 2 |
| Source name |
Pool of all 54 RNAs of this study (samples 1-54)
|
| Organism |
Homo sapiens |
| Characteristics |
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 17.
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®.
Raw data files:
1_Exiqon*: Hy3
0_Exiqon*: Hy5
|
| |
|
| Submission date |
Nov 15, 2012 |
| Last update date |
Dec 31, 2014 |
| Contact name |
Santosh Kumar Patnaik |
| Phone |
716-8458364
|
| Organization name |
Roswell Park Comprehensive Cancer Center
|
| Department |
Thoracic Surgery
|
| Street address |
Elm and Carlton Streets
|
| City |
Buffalo |
| State/province |
NY |
| ZIP/Postal code |
14263 |
| Country |
USA |
| |
|
| Platform ID |
GPL16287 |
| Series (2) |
| GSE42333 |
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 |
| GSE42334 |
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 |
|
