|
| Status |
Public on Feb 15, 2022 |
| Title |
HC-rep1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Circulating CD4+ T Cells_CD4+425(1)_Hy5
|
| Organism |
Homo sapiens |
| Characteristics |
disease state: Healthy
cell type: Circulating CD4+ T Cells
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
|
| Label |
Hy5
|
| Label protocol |
The miRCURY™ Hy3™/Hy5™ Power labeling kit (Exiqon, Vedbaek, Denmark) was used according to the manufacturer’s guideline for miRNA labelling. One microgram of each sample was 3'-end-labeled with Hy3 and Hy5 fluorescent label, respectively, using T4 RNA ligase by the following procedure: RNA in 2.0 μL of water was combined with 1.0 μL of CIP buffer and CIP (Exiqon). The mixture was incubated for 30 min at 37°C, and was terminated by incubation for 5 min at 95°C. Then 3.0 μL of labeling buffer, 1.5 μL of fluorescent label (Hy3 or Hy5), 2.0 μL of DMSO, 2.0 μL of labeling enzyme were added into the mixture. The labeling reaction was incubated for 1 h at 16°C, and terminated by incubation for 15 min at 65°C.
|
| |
|
| Channel 2 |
| Source name |
common reference pool
|
| Organism |
Homo sapiens |
| Characteristics |
sample type: pooled sample including all 14 samples
cell type: Circulating CD4+ T Cells
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
|
| Label |
Hy3
|
| Label protocol |
The miRCURY™ Hy3™/Hy5™ Power labeling kit (Exiqon, Vedbaek, Denmark) was used according to the manufacturer’s guideline for miRNA labelling. One microgram of each sample was 3'-end-labeled with Hy3 and Hy5 fluorescent label, respectively, using T4 RNA ligase by the following procedure: RNA in 2.0 μL of water was combined with 1.0 μL of CIP buffer and CIP (Exiqon). The mixture was incubated for 30 min at 37°C, and was terminated by incubation for 5 min at 95°C. Then 3.0 μL of labeling buffer, 1.5 μL of fluorescent label (Hy3 or Hy5), 2.0 μL of DMSO, 2.0 μL of labeling enzyme were added into the mixture. The labeling reaction was incubated for 1 h at 16°C, and terminated by incubation for 15 min at 65°C.
|
| |
|
| |
| Hybridization protocol |
The Hy3-labeled samples and Hy5-labeled samples were mixed pair-wise and hybridized on the miRCURYTM LNA Array (v.14.0) (Exiqon) according to array manual. The total 25 μL mixture from Hy3TM-labeled samples and Hy5TM-labeled samples with 25 μL hybridization buffer were first denatured for 2 min at 95°C, incubated on ice for 2 min and then hybridized to the microarray for 16–20 h at 56°C in a 12-Bay Hybridization Systems (Hybridization System - Nimblegen Systems, Inc., Madison, WI, USA), which provides an active mixing action and constant incubation temperature to improve hybridization uniformity and enhance signal. Following hybridization, the slides were achieved, washed several times using Wash buffer kit (Exiqon), and finally dried by centrifugation for 5 min at 400 rpm.
|
| Scan protocol |
The slides were scanned using the Axon GenePix 4000B microarray scanner (Axon Instruments, Foster City, CA).
Scanned images were then imported into GenePix Pro 6.0 software (Axon) for grid alignment and data extraction.
|
| Description |
Biological replicate 1 of 6. Healthy Control Group Circulating CD4+ T Cells.
Healthy Control Group Circulating CD4+ T Cells Replicate 1
|
| Data processing |
miRNAs that two channel intensities>0 and SNR>1(or one of channel SNR>2) were chosen for further normalization. Expression data were normalized using the lowess (Locally Weighted Scatter plot Smoothing) regression algorithm (MIDAS, TIGR Microarray Data Analysis System), which can produce within-slide normalization to minimize the intensity-dependent differences between the dyes. Between slides normalization was performed by scale normalization. After normalization, replicated miRNAs were averaged. Differentially expressed miRNAs with statistical significance were identified through Volcano Plot filtering. Hierarchical clustering was performed using MEV software (v4.6, TIGR).
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| |
|
| Submission date |
Feb 14, 2022 |
| Last update date |
Feb 15, 2022 |
| Contact name |
Siwen Wang |
| E-mail(s) |
sven.king@hotmail.com
|
| Organization name |
The First Affiliated Hospital of Sun Yat-sen University
|
| Street address |
58# Zhongshan 2nd Rd
|
| City |
Guangzhou |
| ZIP/Postal code |
510080 |
| Country |
China |
| |
|
| Platform ID |
GPL26002 |
| Series (1) |
| GSE196698 |
MicroRNAs profile in Human Circulating CD4+ T Cells in Patients with Atherosclerosis Obliterans：Healthy persons vs. patients |
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