|
| Status |
Public on Nov 23, 2011 |
| Title |
Control 3 [miRNA] |
| Sample type |
RNA |
| |
|
| Source name |
PBMC
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Blood
infection: none
|
| Treatment protocol |
One half of the cells were subsequently infected with 0.1 MOI of virus particles using standard protocols as described and the remaining half of the culture is maintained under similar conditions and used as control. Seven days post infection (pi), cells were collected and frozen for total RNA isolation.
|
| Growth protocol |
We purchased normal donor blood from the American Red Cross Blood Bank in Pittsburgh using appropriate IRB approval forms from the University of Pittsburgh. PBMCs were isolated by Ficoll-Hypaque gradient centrifugation. Freshly isolated normal donor PBMCs (5×106/mL) were stimulated with 5 µg/ml PHA-P (Sigma, St. Louis, MO) for three days. Cells were washed, divided into two parts and cultured in RPMI medium (GIBCO, CA) containing 10% FBS (Hyclone, Logan, UT), 1% L-glutamine (Cambrex, MD), 1% penicillin-streptomycin (GIBCO, CA), and IL-2 (200 U/mL, Chiron, Emoryville, CA).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using the TRIzol method (InVitrogen) as suggested by the manufacturer. Next, to enhance the sensitivity and detection, we enriched the small RNA using a microRNA isolation kit (SABiosciences) with two separating columns as per the manufacturer's instructions.
|
| Label |
SYBR Green/Rox
|
| Label protocol |
One ug of total RNA enriched for miRNA was reverse transcribed to cDNA using SA Biosciences RT2 miRNA first strand Kit (MA-03). Then the cDNA product along with SA Biosciences RT2 Real-time SYBR Green/Rox PCR master mix (PA-012) was used for detection.
Cycles Duration & Temperature 1 10 minutes at 95°C 40 15 seconds at 95°C -> 40 seconds at 60°C -> 30 seconds at 72°C
|
| |
|
| Hybridization protocol |
n/a
|
| Scan protocol |
n/a
|
| Description |
Uninfected PBMC from donor 3
|
| Data processing |
Raw Ct values obtained after the 7900 HT RT-PCR run were uploaded onto SA Biosciences online analysis tool to generate fold changes for differential expression of miRNA in Infected group i.e. HIV-1 (N=6) over Uninfected group i.e Control group (N=6).
|
| |
|
| Submission date |
Nov 21, 2011 |
| Last update date |
Nov 30, 2011 |
| Contact name |
Pruthvi S. R. Nagilla |
| E-mail(s) |
[email protected]
|
| Phone |
412 624-3062
|
| URL |
http://www.idm.pitt.edu/directory/bios/ayyavoo.asp
|
| Organization name |
University of Pittsburgh
|
| Department |
Infectious Diseases and Microbiology
|
| Street address |
416 Parran Hall, 130 DeSoto Street
|
| City |
Pittsburgh |
| State/province |
PA |
| ZIP/Postal code |
15261 |
| Country |
USA |
| |
|
| Platform ID |
GPL14907 |
| Series (2) |
| GSE33837 |
Comparative Expression Profile of miRNA and mRNA in Primary Peripheral Blood Mononuclear Cells Infected with Human Immunodeficiency Virus (HIV-1) [miRNA] |
| GSE33879 |
Comparative Expression Profile of miRNA and mRNA in Primary Peripheral Blood Mononuclear Cells Infected with Human Immunodeficiency Virus (HIV-1) |
|
