|
| Status |
Public on Nov 26, 2024 |
| Title |
Sample 19 hSPBrepeat |
| Sample type |
RNA |
| |
|
| Source name |
right lung
|
| Organism |
Mus musculus |
| Characteristics |
treatment: AAV-hSPB repeat
Sex: male
strain: CCSP-rtTAtg/(teto)7Sftpb/Sftpb-/- generated on the FVB/N strain
|
| Treatment protocol |
Lungs were flash frozen in liquid nitrogen.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For each lung tissue sample, approximately 30 mg of flash frozen murine lung tissue was placed in 600 µL of lysis buffer (PureLink RNA Mini Kit; Invitrogen, Carlsbad, CA) containing 1% of 2-mercaptoethanol (MP Biomedicals LLC, Santa Ana, CA). Each lung sample was mechanically homogenized on ice for 45 seconds at a speed of 25,000 rpm (maximum speed) using the Fisherbrand Homogenizer 850 (Version 2.1). RNA was extracted according to the manufacturer's instructions (PureLink RNA Mini Kit; Invitrogen), and all samples were stored at -80° C. RNA concentrations and absorbance were measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA). In all samples in this study, the RNA concentrations ranged from 250 to 2000 ng per µL. The A260/A280 ratios (RNA purity) for all samples ranged from 2.09 - 2.17, and the A260/A230 ratios (nucleic acid purity) ranged from 1.3 - 2.4.
|
| Label |
single molecule fluorescent barcodes through nucleotide hybridization
|
| Label protocol |
RNA samples isolated from lung tissue were adjusted to a concentration of 20 ng per µL for gene expression analyses (nCounter).
|
| |
|
| Hybridization protocol |
A total of 100 ng of RNA was hybridized to the Mouse Host Response Panel CodeSet (nCounter Pro MAX/Flex, NanoString). The Mouse Host Response Panel CodeSet includes 773 murine genes plus 12 internal reference genes involved in host susceptibility, the interferon pathway, the innate and adaptive immune response, and homeostasis. The samples were processed by the nCounter Prep Station and run by the Translational Science Laboratory in the Hollings Cancer Center at the Medical University of South Carolina
|
| Scan protocol |
The cartridge containing the probes and the bound RNA were scanned into the Digital Analyzer for image acquisition. The barcodes were counted and analyzed through the nCounter analysis software (nSolver 4.0).
|
| Data processing |
The recommended percentage for successful imaging in nCounter is 75% of 555 or 416 fields of view (FOV). In our samples, FOV ranged from 509 to 554 for an imaging quality control (QC) range of 92% to 99.8%. The binding density QC determines image saturation, and the acceptable range is 0.1 to 2.25. In our samples, binding density ranged from 0.44 to 1.51. The positive control linearity QC is an assessment of the efficacy of hybridization for six synthetic positive controls added to the hybridization reaction. The acceptable calculated R2 value is greater than 0.95. In our samples, the positive control linearity ranged from 0.99 to 1. The limit of detection QC examines the ability to detect the 0.5fM positive control probe. This count should be higher than two times the standard deviation above the mean of the negative control. In our samples the limit of detection QC ranged from 12.28 to 53.17. Normalization, fold changes and P-values were calculated based on criteria provided by Nanostring. ROSALIND® uses the nCounter protocol of dividing counts within a lane by the geometric mean of the normalizer probes from the same lane. Housekeeping probes for normalization were selected based on the geNorm algorithm as in the NormqPCR R library.
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| |
|
| Submission date |
Nov 21, 2024 |
| Last update date |
Nov 26, 2024 |
| Contact name |
Martin Kang |
| Organization name |
Medical University of South Carolina
|
| Department |
Pediatrics
|
| Lab |
Kang Lab
|
| Street address |
173 Ashley Avenue CR405D
|
| City |
Charleston |
| State/province |
SC |
| ZIP/Postal code |
29425 |
| Country |
USA |
| |
|
| Platform ID |
GPL35119 |
| Series (1) |
| GSE282558 |
Novel Immune Response Evasion Strategy to Redose Adeno-Associated Viral Vectors and Prolong Survival in Surfactant Protein-B Deficient Mice |
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