|
| Status |
Public on Sep 15, 2011 |
| Title |
plasma, pre-infection (SIV) PRd2_PB |
| Sample type |
RNA |
| |
|
| Source name |
plasma, pre-infection (SIV)
|
| Organism |
Macaca nemestrina |
| Characteristics |
sample type: plasma
Sex: Male
degree of encephalitis at necropsy: Severe
age: < 5 years
necropsy, days post inoculation: 44
time: pre-infection (SIV)
infection: none
disease state: Severe encephalitis
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from 100 ul plasma with a miRvana isolation kit (Ambion) per the manufacturer's protocol. 5 nM synthetic ath-miR159a was spiked into each sample to correct for technical differences. RNA was eluted in 100 ul nuclease-free water.
|
| Label |
FAM
|
| Label protocol |
Five ul total RNA per sample was reverse-transcribed using Megaplex primers, sets A and B (Applied Biosystems). cDNA was pre-amplified for twelve cycles with Mexaplex Pre-Amp primers, sets A and B (Applied Biosystems), then diluted to 100 ul. Manufacturer's protocol was followed throughout.
|
| |
|
| Hybridization protocol |
n/a
|
| Scan protocol |
n/a
|
| Description |
Total RNA was reverse-transcribed, pre-amplified with miRNA-specific primers, and loaded into TLDA cards (2 per sample). Quantitative real-time PCR was performed for 768 features, 754 of which represent primate miRNAs.
For each of two cards per sample (A and B), nine ul of pre-amplified, diluted sample was mixed with TaqMan reaction mix (Applied Biosystems) and loaded into miRNA TLDA cards (Applied Biosystems) by centrifugation. Cards were sealed, and qRT-PCR was performed with a real-time thermocycler (ABI-7900, Applied Biosystems) per manufacturer's recommendations.
|
| Data processing |
SDS software (Applied Biosystems) was used to collect data.
Threshold intensities were set, visually and individually confirmed, and standardized with RQ Manager (Applied Biosystems). Data were processed using DataAssist (Applied Biosystems) and HTqPCR (R/Bioconductor). In HTqPCR, quantile normalization was employed.
|
| |
|
| Submission date |
Dec 14, 2010 |
| Last update date |
Sep 15, 2011 |
| Contact name |
Kenneth W Witwer |
| E-mail(s) |
[email protected]
|
| Phone |
410-955-9770
|
| Organization name |
Johns Hopkins University School of Medicine
|
| Department |
Molecular and Comparative Pathobiology
|
| Lab |
Retrovirus Lab
|
| Street address |
733 N Broadway, BRB Rm 820
|
| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21205 |
| Country |
USA |
| |
|
| Platform ID |
GPL11316 |
| Series (1) |
| GSE26057 |
miRNA profiling of Macaca nemestrina plasma samples: before SIV infection and during acute infection |
|
