|
| Status |
Public on Nov 01, 2010 |
| Title |
GASneg_sample3 |
| Sample type |
RNA |
| |
|
| Source name |
human pharyngeal/tonsilar swab
|
| Organism |
Homo sapiens |
| Characteristics |
age: <18 years old
culture results: GAS negative
|
| Growth protocol |
Throat swabs containing GAS were maintained at -80°C until nucleic acid extraction was performed.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Throat swabs were incubated in 2ml of lysis buffer (Biomerieux) at 60°C for 30 minutes and then nucleic acids were extracted using the EasyMag automated nucleic acid extraction system (Biomerieux). Genomic DNA was removed using Dnase (Ambion) and verified using PCR.
|
| Label |
Biotin
|
| Label protocol |
One hundred ng of total RNA was amplified using the MessageAmp II-Bacteria Kit (Applied Biosystems). The RNA was labeled with biotin-11-UTP (Perkin Elmer) according to the kit's protocol during the in vitro transcription of aRNA
|
| |
|
| Hybridization protocol |
Microarrays were pre-hybridized for 10 minutes with nuclease-free water at 65°C and then for 30 minutes at 45°C wit hpre-hybridization solution (Combimatrix). Five 5µg of biotin labelled RNA was then fragmented with10X Fragmentation Buffer (Combimatrix) for 20 minutes at 70°C. The fragmented RNA was then hybridized to the microarray using Hybridization solution (Combimatrix) at 45°C for 16 hours.The microarray was then washed 3 times with 6X SSPET (Combimatrix) and then 3 times with 3X SSPET (Combimatrix). The array was then washed 1 time with PBST (Combimatrix). The microarray was then blocked using Blocking Buffer (Combimatrix) for 5 minutes at room temperature and then the Biotin Labeling Solution was added (Combimatrix) and incubated with the microarray for 30 minutes at room temperature. The microarray was then washed 2 times with PBST then TMB Rinse Solution (Combimatrix) beofre being placed into the ElectraSense reader (Combimatrix).
|
| Scan protocol |
Microarrays were inserted into the ElectraSense Microarray Reader and scanned using the ElectraSense application software (Combimatrix). Raw data was stored as an .ECD file.
|
| Description |
NC3
|
| Data processing |
Factory built in negative controls were used for background subtraction. The mean of the lowest 10% of the negative controls was subtracted from all values. To avoid negative numbers, 2 standard deviations of the mean was used to replace all negative numbers. All samples were normalized using a median quantile method performed with ProbeWeaver software (Combimatrix Corporation). The Feature # 0 in the raw file corresponds with ID #1 for the GPL10562 full array layout.
|
| |
|
| Submission date |
Jun 18, 2010 |
| Last update date |
Aug 24, 2010 |
| Contact name |
Jeffrey Livezey |
| E-mail(s) |
[email protected]
|
| Organization name |
Brooke Army Medical Center
|
| Department |
Clinical Investigation
|
| Street address |
3400 Rawley E Chambers Ave
|
| City |
San Antonio |
| State/province |
TX |
| ZIP/Postal code |
78247 |
| Country |
USA |
| |
|
| Platform ID |
GPL10562 |
| Series (1) |
| GSE22436 |
Group A Streptococcus Gene Expression in Humans with Pharyngitis Using a Microarray |
|
