|
Status |
Public on Mar 31, 2018 |
Title |
Negative Control 2 Macs |
Sample type |
RNA |
|
|
Channel 1 |
Source name |
Negative Control 2 Macs
|
Organism |
Homo sapiens |
Characteristics |
cell type: Primary human Macrophages transfected with: control mimic passage: None
|
Treatment protocol |
Transient transfections of miR-H1, miR-K12-3-3p or control mimic were performed using Lipofectaine 2000 reagent (Life Technologies, San Diego, CA) according to manufacturer’s instructions.
|
Growth protocol |
Human gingival keratinocytes: Primary HGEC were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA). These cells were isolated from human oral mucosa and cryopreserved on passage one culture. Cells were cultured using Keratinocyte Medium (LifeLine Cell Technology, Friedrich, MD) which contains basal medium, growth supplements (hormones, growth factors and proteins) and penicillin/streptomycin. Macrophages: Monocytes were isolated from freshly prepared buffy coats collected from healthy donors (n ≥ 3, Sylvan N. Goldman Oklahoma Blood Institute, Oklahoma City, OK, USA) by density gradient centrifugation and magnetic bead sorting as previously described (Self-Fordham et al., 2015; Naqvi et al., 2015, 2016).
|
Extracted molecule |
total RNA |
Extraction protocol |
After 36 hours, cells were harvested for RNA isolation using miRNeasy kit according to manufacturer's protocol. The reference RNA is a pool of total RNA from 20 different normal, human tissues. This is commercially available as FirstChoice(r) Human Total RNA Survey Panel at ThermoFisher Catalog Number AM6000.
|
Label |
Hy3
|
Label protocol |
Total RNA (225 ng) was labeled by use ofusing the miRCURY LNA microRNA Hi-Power Labeling Kit Hy3/Hy5 following the procedures described by the manufacturer.
|
|
|
Channel 2 |
Source name |
Reference RNA
|
Organism |
Homo sapiens |
Characteristics |
control type: pool of total RNA from 20 different normal, human tissues.
|
Treatment protocol |
Transient transfections of miR-H1, miR-K12-3-3p or control mimic were performed using Lipofectaine 2000 reagent (Life Technologies, San Diego, CA) according to manufacturer’s instructions.
|
Growth protocol |
Human gingival keratinocytes: Primary HGEC were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA). These cells were isolated from human oral mucosa and cryopreserved on passage one culture. Cells were cultured using Keratinocyte Medium (LifeLine Cell Technology, Friedrich, MD) which contains basal medium, growth supplements (hormones, growth factors and proteins) and penicillin/streptomycin. Macrophages: Monocytes were isolated from freshly prepared buffy coats collected from healthy donors (n ≥ 3, Sylvan N. Goldman Oklahoma Blood Institute, Oklahoma City, OK, USA) by density gradient centrifugation and magnetic bead sorting as previously described (Self-Fordham et al., 2015; Naqvi et al., 2015, 2016).
|
Extracted molecule |
total RNA |
Extraction protocol |
After 36 hours, cells were harvested for RNA isolation using miRNeasy kit according to manufacturer's protocol. The reference RNA is a pool of total RNA from 20 different normal, human tissues. This is commercially available as FirstChoice(r) Human Total RNA Survey Panel at ThermoFisher Catalog Number AM6000.
|
Label |
Hy5
|
Label protocol |
Total RNA (225 ng) was labeled by use ofusing the miRCURY LNA microRNA Hi-Power Labeling Kit Hy3/Hy5 following the procedures described by the manufacturer.
|
|
|
|
Hybridization protocol |
RNA was hybridized onto miRCURY LNA microRNA (7th Gen; miRBase v19) arrays following the procedures described by the manufacturer.
|
Scan protocol |
After hybridization the microarray slides were scanned and stored in an ozone free environment (ozone level below 2.0 ppb) in order to prevent potential bleaching of the fluorescent dyes. The miRCURY LNA™ microRNA Array slides were scanned using the Agilent G2565BA Microarray Scanner System (Agilent Technologies, Inc., USA) and the image analysis was carried out using the ImaGene® 9 (miRCURY LNA™ microRNA Array Analysis Software, Exiqon, Denmark).
|
Description |
Sample 30
|
Data processing |
Data normalization were performed by Exiqon using Quantile normalization. The quantified signals were background corrected (Normexp with offset value 10, see Ritchie et al. 2007) and normalized using the global Lowess (LOcally WEighted Scatterplot Smoothing) regression algorithm. Initial analysis was performed by Exiqon using R/bioconductor, primarily by use of the limma package (Exiqon). Expression analysis of variance over time was performed with P values adjusted using the Benjamini-Hochberg method and identified genes subjected to the Tukey’s "honest significant difference" test. The file "normalized_data.txt" contains the normalized Hy3 values. This file is available on the series record.
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|
|
Submission date |
Dec 04, 2017 |
Last update date |
Mar 31, 2018 |
Contact name |
Afsar Naqvi |
E-mail(s) |
afsarraz@uic.edu
|
Organization name |
University of Illinois at Chicago
|
Department |
Periodontics
|
Lab |
Mucosal Immunology Lab
|
Street address |
801 S Paulina, Room 561B
|
City |
Chicago |
State/province |
IL |
ZIP/Postal code |
60612 |
Country |
USA |
|
|
Platform ID |
GPL17107 |
Series (1) |
GSE107674 |
Viral miRNAs alter cellular miRNAs profiles |
|