|
| Status |
Public on Mar 09, 2016 |
| Title |
MWCNT Exposed Worker-5 (Scientist) |
| Sample type |
RNA |
| |
|
| Source name |
Blood
|
| Organism |
Homo sapiens |
| Characteristics |
gender: Male
tissue: Whole Blood
age: 63
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from whole blood using the PAXgene™ Blood RNA System Kit following manufacturer's guidelines. Briefly, the samples were removed from -80°C and incubated at room temperature for 2 hours to ensure complete lysis. Following lysis the tubes were centrifuged for 10 min at 5,000 × g, the supernatant decanted and 500 μL of RNase-free water added to the pellet. After washing with 500 μl RNase-free water, the pellet was dissolved in 350 μl resuspension buffer and incubated with 300 μl binding buffer and 40 μl proteinase K for 10 min at 55°C in a shaker-incubator. The lysate was transferred into a PAXgene shredder spin column and centrifuged (at 18,000 g for 3 min). The flow-through fraction was mixed with 350 μl ethanol and transferred to a PAXgene RNA spin column. After washing the column with washing buffer 1, samples were incubated with 10 μl of DNase I for 15 min. PAXgene RNA spin columns were washed with washing buffer and RNA eluted with 40 μl elution buffer. The RNA yield was estimated by measuring absorbance at 260 nm in a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). RNA purity was calculated from the ratio of absorbance at 260 nm and 280 nm, and RNA integrity was assessed by standard denaturing agarose gel electrophoresis.
|
| Label |
Cy3
|
| Label protocol |
total RNA after removal of rRNA (mRNA-ONLY™ Eukaryotic mRNA Isolation Kit, Epicentre) was amplified and transcribed into fluorescent cRNA along the entire length of the transcripts without 3’ bias utilizing a random priming method. The labeled cRNAs were purified by RNeasy Mini Kit (Qiagen). The concentration and specific activity of the labeled cRNAs (pmol Cy3/μg cRNA) were measured by NanoDrop ND-1000.
|
| |
|
| Hybridization protocol |
The labeled cRNAs were hybridized onto the Human LncRNA Array v3.0 (8 x 60K, Arraystar). Breifly, 1 μg of each labeled cRNA was fragmented by adding 5 μl 10 × Blocking Agent and 1 μl of 25 × Fragmentation Buffer, then heated the mixture at 60 °C for 30 min, finally 25 μl 2 × GE Hybridization buffer was added to dilute the labeled cRNA. 50 μl of hybridization solution was dispensed into the gasket slide and assembled to the LncRNA expression microarray slide. The slides were incubated for 17 hours at 65°C in an Agilent Hybridization Oven.
|
| Scan protocol |
After having washed the slides, the arrays were scanned by the Agilent Scanner G2505C. Agilent Feature Extraction software (version 11.0.1.1) was used to analyze the acquired array images.
|
| Description |
whole blood lncRNA and mRNA expression in MWCNT-exposed workers
|
| Data processing |
Quantile normalization and subsequent data processing were performed using the GeneSpring GX v11.5.1 software package (Agilent Technologies). After quantile normalization of the raw data, LncRNAs and mRNAs that at least 5 out of 15 samples have flags in Present or Marginal (“All Targets Value”) were chosen for further data analysis. Differentially expressed LncRNAs and mRNAs with statistical significance were identified through Volcano Plot filtering between two groups.
|
| |
|
| Submission date |
Oct 07, 2015 |
| Last update date |
Mar 09, 2016 |
| Contact name |
Anna A Shvedova |
| E-mail(s) |
ats1@cdc.gov
|
| Organization name |
CDC/NIOSH
|
| Department |
HELD
|
| Lab |
EAB
|
| Street address |
1095 willowdale Rd
|
| City |
morgantown |
| State/province |
WV |
| ZIP/Postal code |
26508 |
| Country |
USA |
| |
|
| Platform ID |
GPL16956 |
| Series (1) |
| GSE73830 |
Whole Blood long non-coding RNA and mRNA Expression Profiles in Humans Exposed to Multi-walled Carbon Nanotubes |
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