|
| Status |
Public on Jan 11, 2022 |
| Title |
276M |
| Sample type |
RNA |
| |
|
| Source name |
Myometrium #276
|
| Organism |
Homo sapiens |
| Characteristics |
race: Caucasian
genotype: MED12 Mutant
|
| Treatment protocol |
Portions of uterine leiomyomas and paired myometrium were obtained from patients who were not taking any hormonal medications for at least 3 months prior to surgery at Harbor-UCLA Medical Center.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from leiomyoma and matched myometrium using Trizol. RNA concentration and integrity was determined using a Nanodrop 2000c spectrophotometer and Agilent 2100 Bioanalyzer.
|
| Label |
Cy3
|
| Label protocol |
An efficient and robust linear amplification method to produce fluorescently labeled target cRNA for sensitive detection of transcripts at even low copy numbers. Sample labeling and array hybridization were performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technology) with minor modifications. Briefly, mRNA was purified from total RNA after removal of rRNA (mRNA-ONLY™ Eukaryotic mRNA Isolation Kit, Epicentre). Then, each sample was amplified and transcribed into fluorescent cRNA along the entire length of the transcripts without 3' bias utilizing a random priming method (Arraystar Flash RNA Labeling Kit, Arraystar). The 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.
|
| |
|
| 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 |
Myometrium #276
|
| 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 with using the GeneSpring GX v12.1 software package (Agilent Technologies), and adjusting batch effects in expression data using empirical Bayes methods. SE-LncRNAs and mRNAs that at least 2 out of 16 samples have flags in Present or Marginal (“All Targets Value”) were chosen for further data analysis. Differentially expressed SE-LncRNAs and mRNAs with statistical significance between the two groups were identified through Volcano Plot filtering. Differentially expressed LncRNAs and mRNAs between the two samples 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. Hierarchical Clustering and combined analysis were performed using in-house scripts.
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| |
|
| Submission date |
Jan 09, 2022 |
| Last update date |
Jan 12, 2022 |
| Contact name |
Omid Khorram |
| Organization name |
The lundquist Institute at UCLA medical center
|
| Street address |
1124 w carson st
|
| City |
torrance |
| State/province |
CA |
| ZIP/Postal code |
90502 |
| Country |
USA |
| |
|
| Platform ID |
GPL24592 |
| Series (1) |
| GSE193320 |
Differential Expression of Super-enhancer-associated Long Non-coding RNAs in Uterine Leiomyomas |
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