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| Status |
Public on Jul 26, 2019 |
| Title |
2cell HMVEC 6hr #2 |
| Sample type |
RNA |
| |
|
| Source name |
HMVEC 2cell 1.2ug/ml MWCNT 6hr
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HMVEC
culture: coculture
|
| Treatment protocol |
For cell culture exposures, MWCNT were prepared in DM. Transmission electron micrographs of MWCNT dispersed in DM determined that DM promoted significant dispersion of MWCNT. MWCNT were prepared in DM, followed by indirect sonication at 4°C for 5 min (Hielscher ultrasonic processor, UIS259L) at amplitude 100% and cycle 1. Following indirect sonication, the suspension was directly sonicated for 5 min at 5W output and 10% duty cycle (Branson Sonifier 450). The stock solution (0.5 mg/mL) of MWCNT was kept at 4°C and used within 2–3 weeks. Prior to cell culture experiments, the MWCNT stock solution was directly sonicated for 1 min at 5W output and 10% duty cycle.
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| Growth protocol |
For monoculture exposures, SAEC and HMVEC were plated directly into 100 mm cell culture dishes (Corning, Tewksbury, MA; growth area: 55 cm2), allowed to form intact barriers for 72 h, serum starved overnight, and exposed directly to MWCNT at a concentration of 1.2 µg/mL in 10 mL of their respective media for either 6 or 24 h. DM for 24 h was used as a negative control. Six biological replicates of each SAEC and HMVEC monoculture exposure condition were collected for microarray analysis. Both SAEC and HMVEC in monoculture have been shown to interact with MWCNT (Pacurari et al. 2012; Snyder-Talkington et al. 2013b). For coculture exposures, SAEC and HMVEC were allowed to form intact epithelial and endothelial barriers for 72 h, serum starved overnight, and SAEC were exposed to MWCNT at a concentration of 1.2 µg/mL in 10 mL of SAEC media for either 6 or 24 h. DM for 24 h was used as a negative control. HMVEC in the coculture system were not directly exposed to MWCNT, and MWCNT are not apparent in HMVEC transmission electron microscopy preparations following SAEC exposure in coculture (Snyder-Talkington et al. 2013a).Six biological replicates of each SAEC and HMVEC coculture at each exposure condition were collected for microarray analysis. The total number of monoculture and coculture samples for microarray analysis was 72.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from SAEC and HMVEC using RNAprotect Cell Reagent and an RNeasy Mini Kit from Qiagen according to the manufacturer’s protocol (Qiagen, Valencia, CA). RNA concentrations were determined using a NanoDrop 1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE), and RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Oyster-550
|
| Label protocol |
The RNA samples were DNAse digested and low-molecular weight (LMW) RNA was isolated by ultrafiltration through Nanosep 100K columns (Pall Corporation) and subsequent purification using the RNeasy MinElute Clean-Up Kit (Qiagen). The LMW RNA samples were 3’-end labeled with Oyster-550 fluorescent dye using the Flash Tag RNA labeling Kit (Genisphere).
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| |
|
| Hybridization protocol |
Labeled LMW RNA samples were hybridized to the MicroRNA microarrays according to conditions recommended in the Flash Tag RNA labeling Kit manual.
|
| Scan protocol |
The microarrays were scanned on an Axon Genepix 4000B scanner, and data was extracted from images using GenePix V4.1 software.
|
| Description |
2012-05-24_19706189_650PMT_0532.txt
Array # (Block ID): C
|
| Data processing |
Data for 4 arrays were extracted from a single microarray image into a single text file using GenePix version 4.1 software. The raw text files were split in to four subfiles based on block assignment, namely blocks 1-8 contained data for array A, blocks 9-16 contained data for array B, blocks 17-24 contained data for array C, and blocks 25-32 contained data for array D. Spot intensities were obtained for the 8816 features on each microarray by subtracting the median local background from the median local foreground for each spot. The detection threshold (T) for each microarray was computed by summing five times the standard deviation of the background signal and the 10% trim mean of the negative control spots. The spot intensities and T were transformed by taking the log (base 2) of each value; and all further calculations were performed with the log-transformed values. The spot intensities for the 8760 features associated with an oligonucleotide probe were averaged per probe in order to obtain the mean of the triplicate spots for each probe (i.e. probe intensities). The normalization factor (N) for each microarray was calculated as the 20% trim mean of the average human probes spot intensities above threshold in all samples. The probe intensities were then normalized, by subtracting N from each spot intensity and adding back the grand mean of N across all microarrays. The data was filtered to retain values for the 1718 human microRNA probes on the microarray minus any probes showing an overall saturation of signal.
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| |
|
| Submission date |
May 13, 2019 |
| Last update date |
Jul 26, 2019 |
| Contact name |
Nancy Lan Guo |
| E-mail(s) |
lguo@hsc.wvu.edu
|
| Phone |
3042936455
|
| Organization name |
West Virginia University
|
| Department |
Occupational and Environmental Health Sciences
|
| Lab |
Mary Babb Randolph Cancer Center/Guo Laboratory
|
| Street address |
2816 Health Sciences Center
|
| City |
Morgatown |
| State/province |
WV |
| ZIP/Postal code |
26506-9300 |
| Country |
USA |
| |
|
| Platform ID |
GPL26659 |
| Series (2) |
| GSE131121 |
coculture SAEC and HMVEC exposed to MWCNT |
| GSE131123 |
miRNA profiling of MWCNT exposed mice |
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