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Status |
Public on May 10, 2011 |
Title |
Healthy Donor 1 |
Sample type |
RNA |
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Source name |
peripheral blood mononuclear cells
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Organism |
Homo sapiens |
Characteristics |
disease state: Healthy non infected individual: donor 1 tissue: Peripheral venous blood cell type: peripheral blood mononuclear cells gender: Female age: 32y
|
Treatment protocol |
Peripheral venous blood (10 mL) was drawn from each subject and PBMCs were isolated on Ficoll gradients (GE Health Care, Little Chalfont, U.K); PBMCs were immediately mixed with the miR VanaTM miRNA isolation kit Lysis Buffer (Ambion Inc .Austin, Tx) and frozen at −80°C until RNA was extracted.
|
Extracted molecule |
total RNA |
Extraction protocol |
Total RNA was extracted using the miR VanaTM miRNA isolation kit (Ambion Inc .Austin, Tx) according to the manufacturer’s instructions. RNA quantity and quality was assessed using the Nanodrop 2000 (Thermo Fisher Scientific, Mass) and Agilent 2100 bioanalyzer systems (Agilent Technologies). Samples with a RNA Integrity Number above 7 were used in the study.
|
Label |
Cy3
|
Label protocol |
100 ng total RNA (50 ng /ul )were first treat with phosohatase (TakaRa p/n 2250A)at 37 ºC for 30 min .After adding DMSO(Sigma p/n D8418) , samples were heat at 100 ºC for 5-10 min and incubate in ice immediately .Assemble labeling reaction with pCp-Cy3 were then performed by using T4 RNA Ligase at 16ºC for 2h. labeled RNA were then purified by using the Micro Bio spin 6 columns(Bio-Rad p/n 732-6221).
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Hybridization protocol |
Cy3-labelled RNAs were heat at 100 °C for 5 minutes in a reaction volume containing 1X GE Blocking Agent and 1X Hi-RPM Hybridization Buffer following the manufacturers instructions. Samples were transfer to ice water baths for 5 min after and then hybridized to Agilent-021827 Human miRNA Microarray (V3) (miRBase release 12.0 miRNA ID version) for 20 hours at 55°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37 °C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
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Scan protocol |
Microarray slides were scanned by using the XDR Scan (PMT100, PMT5).(Scan Area 61 x 21.6 mm ; Scan resolution 5 um; Single pass; eXtended Dynamic range selected ; Green Dye channel ; Green PMT XDR Hi 100% , XDR Lo 5% )
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Description |
Healthy non infected donor 1
|
Data processing |
The scanned images were analyzed with Feature Extraction Software 9.5.3 (Agilent) using default parameters (protocol miRNA-v1_95_May07) to obtain background subtracted and spatially detrended Processed Signal intensities. The signal (after background subtraction) was exported directly into the GeneSpring GX9.0 software (Agilent Technologies) for quantile normalization.
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Submission date |
May 10, 2011 |
Last update date |
May 10, 2011 |
Contact name |
qian gao |
E-mail(s) |
qgao99@yahoo.com
|
Organization name |
Fudan University
|
Department |
Shanghai Medical School
|
Lab |
Key Laboratory of Medical Molecular Virology
|
Street address |
138, Yi xue yuan Road
|
City |
Shanghai |
ZIP/Postal code |
200032 |
Country |
China |
|
|
Platform ID |
GPL10850 |
Series (1) |
GSE29190 |
Comparative miRNA Expression Profiles in Individuals with Latent and Active Tuberculosis |
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