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| Status |
Public on Jul 31, 2017 |
| Title |
ExVivoBlood_paroxysmal atrial fibrillation_peripheral blood_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Peripheral Blood_paroxysmal atrial fibrillation
|
| Organism |
Homo sapiens |
| Characteristics |
subject status: paroxysmal atrial fibrillation (pAF) patient
gender: Male
age: 67y
tissue: Peripheral Blood (PB)
cell type: leukocytes
|
| Extracted molecule |
total RNA |
| Extraction protocol |
According to the manufacturer’s instructions, leukocytes were separated from whole blood using Red Blood Cell Lysis Buffer (TBD, Tianjin, China). Then RNA was extracted from leukocytes by Trizol reagent (Invitrogen, Carlsbad, CA, USA). A NanoDrop ND-1000 spectrophotometer was used for evaluating RNA quantity and quality. RNA integrity was assessed by standard denaturing agarose gel electrophoresis. Total RNA of the sample was used for lncRNA microarray analysis and the remaining sample was used for further RT-PCR validation.
|
| Label |
Cy3
|
| Label protocol |
mRNA was purified from total RNA after removal of rRNA (mRNA-ONLY™ Eukaryotic mRNA Isolation Kit, Epicentre). Then, 1ug RNA was amplified and transcribed into fluorescent cRNA along the entire length of the transcripts without 3’ bias utilizing a random priming method. The Cyanine-3 (Cy3) labeled cRNAs were purified by RNeasy Mini Kit (Qiagen). The concentration and specific activity of the labeled cRNAs (pmol Cy3/μg cRNA) were measured by NanoDrop ND-1000 Spectrophotometer.
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| |
|
| Hybridization protocol |
1 μg of each labeled cRNA was fragmented by adding 5 μl 10 × Blocking Agent and 1 μl of 25 × Fragmentation Buffer, then heated the mixture at 60 °C for 30 min, finally 25 μl 2 × GE Hybridization buffer was added to dilute the labeled cRNA. 50 μl of hybridization solution was dispensed into the gasket slide and assembled to the LncRNA expression microarray slide. The slides were incubated for 17 hours at 65°C in an Agilent Hybridization Oven.
|
| Scan protocol |
The hybridized arrays were washed, fixed and scanned with using the Agilent DNA Microarray Scanner (part number G2505C).
|
| Description |
W4
lncRNAs and mRNAs expression profiles of peripheral blood leukocytes in paroxysmal atrial fibrillation patient
|
| Data processing |
Agilent Feature Extraction software (version 11.0.1.1) was used to analyze acquired array images. Quantile normalization and subsequent data processing were performed using the GeneSpring GX v11.5.1 software package (Agilent Technologies). After quantile normalization of the raw data, LncRNAs and mRNAs that at least 3 out of 9 samples have flags in Present or Marginal (“All Targets Value”) were chosen for further data analysis (37,069 ID_REFs). Differentially expressed LncRNAs and mRNAs with statistical significance between two groups were identified through Volcano Plot filtering and differentially expressed LncRNAs/mRNAs were identified through Fold Change filtering. Pathway analysis and GO analysis were applied to determine the roles of these differentially expressed mRNAs played in these biological pathways or GO terms. Finally, Hierarchical Clustering was performed to show the distinguishable LncRNAs and mRNAs expression pattern among samples.
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| |
|
| Submission date |
Nov 17, 2015 |
| Last update date |
Jul 31, 2017 |
| Contact name |
Ying Su |
| Organization name |
Capital Medical University affiliated Beijing Shijitan Hospital
|
| Street address |
Tieyi Rd. No.10, Yangfangdian, Haidian district
|
| City |
Beijing |
| ZIP/Postal code |
100038 |
| Country |
China |
| |
|
| Platform ID |
GPL16956 |
| Series (1) |
| GSE75092 |
The long noncoding RNA expression profiles of paroxysmal atrial fibrillation identified by microarray analysis |
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