|
| Status |
Public on Nov 12, 2018 |
| Title |
old-RSV-d5-#55 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Lung
|
| Organism |
Mus musculus |
| Characteristics |
age: old
infection: RSV
day: 5
animal number: 55
|
| Treatment protocol |
Mice were inoculated intranasally with 50 µl containing an infectious dose of 10^6 TCID50 RSV type A2 or mock as control, under anesthesia by isoflurane. At day 2 and 5 after inoculation, mice were anesthetized with Ketamine/Xylazine, bled and sacrificed to collect lungs. Lungs were perfused with PBS to eliminate blood cells prior to collection of the tissue, immersed in RNAlater RNA stabilization agent (Ambion) and subsequently stored at -80ᵒC until further processing.
|
| Growth protocol |
We used adult (6-8 weeks) and old (62-72 weeks) C57BL/6JRccHsd mice. Mice were kept in a 12-hour light/dark cycle under SPF and temperature-controlled conditions, and were fed ad libitum.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from homogenized lung tissue using the Qiagen RNeasy kit. RNA concentrations were measured using a NanoDrop spectrophotometer and RNA quality was measured using a Agilent Bioanalyzer.
|
| Label |
Cy3
|
| Label protocol |
Per sample, 500 ng of RNA was amplified and labeled using the Agilent QuickAmp kit. Samples were labeled with Cy3 and the common reference (made by pooling equimolar amounts of antisense RNA from individual samples) was labeled with Cy5.
|
| |
|
| Channel 2 |
| Source name |
Lung
|
| Organism |
Mus musculus |
| Characteristics |
sample type: common reference
|
| Treatment protocol |
Mice were inoculated intranasally with 50 µl containing an infectious dose of 10^6 TCID50 RSV type A2 or mock as control, under anesthesia by isoflurane. At day 2 and 5 after inoculation, mice were anesthetized with Ketamine/Xylazine, bled and sacrificed to collect lungs. Lungs were perfused with PBS to eliminate blood cells prior to collection of the tissue, immersed in RNAlater RNA stabilization agent (Ambion) and subsequently stored at -80ᵒC until further processing.
|
| Growth protocol |
We used adult (6-8 weeks) and old (62-72 weeks) C57BL/6JRccHsd mice. Mice were kept in a 12-hour light/dark cycle under SPF and temperature-controlled conditions, and were fed ad libitum.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from homogenized lung tissue using the Qiagen RNeasy kit. RNA concentrations were measured using a NanoDrop spectrophotometer and RNA quality was measured using a Agilent Bioanalyzer.
|
| Label |
Cy5
|
| Label protocol |
Per sample, 500 ng of RNA was amplified and labeled using the Agilent QuickAmp kit. Samples were labeled with Cy3 and the common reference (made by pooling equimolar amounts of antisense RNA from individual samples) was labeled with Cy5.
|
| |
|
| |
| Hybridization protocol |
Samples were hybridized to NimbleGen 12x135 k Mus musculus microarrays, containing probes for 44,170 genes with 3 spots per target probe. Each microarray corresponded to labeled RNA from one individual mouse.
|
| Scan protocol |
Slides were scanned in an ozone-free room with a Agilent G2565CA microarray scanner. Feature extraction was performed with NimbleScan v2.5 (Roche NimbleGen) resulting in a table containing individual probe signal intensities for both dyes.
|
| Description |
lung RNA
common reference
FTR files: Cy3 #1, Cy5 #2
|
| Data processing |
Data for gene-coding probes were normalized in R by using a four step approach consisting of: (1) natural log-transformation, (2) quantile normalization of all scans, (3) correcting the sample spot signal for its corresponding reference spot signal and (4) averaging data from replicate probe spots. This resulted in normalized data for 44170 probes, which was used for further statistical analysis in R and Microsoft Excel.
|
| |
|
| Submission date |
Mar 16, 2018 |
| Last update date |
Nov 12, 2018 |
| Contact name |
Jeroen Pennings |
| E-mail(s) |
Jeroen.Pennings@rivm.nl
|
| Phone |
+31 88 689 2214
|
| Organization name |
Natl. Inst. Public Health & Environment
|
| Street address |
A. van Leeuwenhoeklaan 9
|
| City |
Bilthoven |
| ZIP/Postal code |
3721MA |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL10192 |
| Series (1) |
| GSE111947 |
Lung response to Respiratory Syncytial Virus Infection in Adult and Old Mice |
|
