|
| Status |
Public on Feb 06, 2021 |
| Title |
244 Nasal/Oral |
| Sample type |
other |
| |
|
| Channel 1 |
| Source name |
Nasal and Oral tissue
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Nasal and Oral tissue
gender: Female
age: 72yrs
molecule: RNA and DNA
|
| Growth protocol |
Reference sample NR52285 was obtained from BEI resource ATCC and 15 patients samples (oropharyngeal and nasopharyngeal swabs) were obtained from Hospital of University of Pennsylvania, eluted in viral transport media (VTM), then heat inactivated at 56°C for 30 minutes prior to transfer to the laboratory where the DNA and RNA were extracted.
|
| Extracted molecule |
other |
| Extraction protocol |
The swab samples and the blank control samples were were micro-centrifuged at 4°C at 15,000rpms for 20 minutes which will collect all cells, virions and other microbial agents eluted in the VTM. The pelleted samples were then resuspended for simultaneous DNA and RNA extraction (AllPrep DNA/RNA FFPE Kit, Qiagen, Hilden, Germany). The quality of extracted nucleic acids was determined by A260/280 measurements. WTA were performed using the TransPlex Complete Whole Transcriptome Amplification Kit (Sigma-Aldrich, St. Louis, MO) for each specimen using extracted DNA and RNA.Reference control human DNA and RNA was extracted from the human B cell line, BJAB (obtained from ATCC, Manassas, VA)
|
| Label |
Cy3
|
| Label protocol |
1μg of the amplified products from the patient and reference genomes were labelled separately with Cy3 and Cy5, respectively (SureTag labeling kit, Agilent Technologies, Santa Clara, CA). All labeled controls and experimental specimens were purified and hybridized to the PathoChIP microarrays
|
| |
|
| Channel 2 |
| Source name |
BJAB
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: BJAB
molecule: RNA and DNA
|
| Growth protocol |
Reference sample NR52285 was obtained from BEI resource ATCC and 15 patients samples (oropharyngeal and nasopharyngeal swabs) were obtained from Hospital of University of Pennsylvania, eluted in viral transport media (VTM), then heat inactivated at 56°C for 30 minutes prior to transfer to the laboratory where the DNA and RNA were extracted.
|
| Extracted molecule |
other |
| Extraction protocol |
The swab samples and the blank control samples were were micro-centrifuged at 4°C at 15,000rpms for 20 minutes which will collect all cells, virions and other microbial agents eluted in the VTM. The pelleted samples were then resuspended for simultaneous DNA and RNA extraction (AllPrep DNA/RNA FFPE Kit, Qiagen, Hilden, Germany). The quality of extracted nucleic acids was determined by A260/280 measurements. WTA were performed using the TransPlex Complete Whole Transcriptome Amplification Kit (Sigma-Aldrich, St. Louis, MO) for each specimen using extracted DNA and RNA.Reference control human DNA and RNA was extracted from the human B cell line, BJAB (obtained from ATCC, Manassas, VA)
|
| Label |
Cy5
|
| Label protocol |
1μg of the amplified products from the patient and reference genomes were labelled separately with Cy3 and Cy5, respectively (SureTag labeling kit, Agilent Technologies, Santa Clara, CA). All labeled controls and experimental specimens were purified and hybridized to the PathoChIP microarrays
|
| |
|
| |
| Hybridization protocol |
For each PathoChIP array, a Cy3 labeled specimen and a Cy5 labeled reference were combined and hybridized together in constant rotation at 65°C for 12 hours. The slides were then washed and scanned for visualization using an Agilent SureScan G4900DA array scanner.
|
| Scan protocol |
Agilent SureScan G4900DA array scanner was used to scan the microarrays.
|
| Data processing |
Agilent Feature Extraction software was used for extraction of the raw green and red signal data from the microarray images captured by the G400DA scanner.
A scale factor, which was inferred from HRP, was set as 0.3 to control the effect from red to green signal. Specifically, we multiply the log2 red signal by the scale factor to get the normalized background signal which was then subtracted from the log2 green signal to get the final normalized data. The normalized value of a probe is called hybridization signal intensity (HSI) which indicates the abundance of the species of the probe in the tested sample.
|
| |
|
| Submission date |
Feb 05, 2021 |
| Last update date |
Feb 06, 2021 |
| Contact name |
Erle S Robertson |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Pennsylvania
|
| Department |
Department of Otorhinolaryngology
|
| Street address |
3610 Hamilton Walk Johnson Pavilion
|
| City |
Philadelphia |
| State/province |
Pennsylvania |
| ZIP/Postal code |
19104 |
| Country |
USA |
| |
|
| Platform ID |
GPL29680 |
| Series (1) |
| GSE166281 |
SARS-CoV-2 Oropharyngeal and Nasopharyngeal samples |
|
