|
| Status |
Public on Mar 23, 2012 |
| Title |
EoE patient #2_after treatment [miRNA] |
| Sample type |
RNA |
| |
|
| Source name |
EoE patient_after treatment
|
| Organism |
Homo sapiens |
| Characteristics |
age range: Pediatric patient
clinical procedure: Biopsy
disease status: After glucocorticoid steroid treatment
tissue: epithelium
histology procedure: Formalin-fixed paraffin-embedded
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Tissue sections (10mm) were cut and mounted onto plain glass slides. If the biopsy section contained only epithelium, the sections were scraped from the slides and RNA was extracted using the RecoverAll Total Nucleic Acid Extraction kit for FFPE tissues (Ambion). If biopsy sections contained sub-epithelium, the sections were deparaffinized, stained, dehydrated through graded alcohols using the Paradise FFPE reagent System (Applied Biosystems, Foster City, CA) and subjected to LCM within 2 hr of deparaffinization. About 20000 epithelial cells were captured on LCM Macro CapSure caps (Applied Biosystems) using the Arcturus XT LCM instrument (Applied Biosystems). Total RNA was extracted from the caps also using the RecoverAll kit as above, and subjected to analysis by the Agilent Bioanalyzer using an RNA 6000 Nano or Pico LabChip (Agilent Technologies)
|
| Label |
FAM
|
| Label protocol |
Five nanograms of total RNA was reverse-transcribed using the Taqman MicroRNA Reverse Transcription Kit and the Megaplex RT primer Human Pool A (Applied Biosystems). The reverse-transcribed cDNA was then pre-amplified in 12 cycles of PCR using Taqman PreAmp Master Mix and the Megaplex PreAmp primers, Human Pool A (Applied Biosystems). The cDNA’s were then diluted and loaded on to a Taqman Human miRNA Array card A (Applied Biosystems), which contains probes for 377 distinct miRNAs. The Array cards were run on an ABI HT7900 qPCR instrument.
|
| |
|
| Hybridization protocol |
n/a
|
| Scan protocol |
n/a
|
| Description |
Fresh bipspies were fixed right away and porcessed within the same day.
EE-2A
|
| Data processing |
The normalization and all the data analysis were performed according to the manufacturers instructions of ABI HT7900.
Original Ct values were normalized based on mammalian U6 as housekeeping gene control.
Target gene signals (delta Ct) normalized to housekeeping genes; delta Ct = (Ct_Target − Ct_HKG)
The folder change of each miRNA is calculated using 2^-delta delta Ct (before/after treatment ratios).
|
| |
|
| Submission date |
Mar 22, 2012 |
| Last update date |
Mar 23, 2012 |
| Contact name |
Shaolei Lu |
| E-mail(s) |
slu@lifespan.org
|
| Phone |
401-444-5709
|
| Organization name |
Rhode Island Hospital
|
| Department |
Pathology
|
| Street address |
593 Eddy Steet
|
| City |
Providence |
| State/province |
Rhode Island |
| ZIP/Postal code |
02903 |
| Country |
USA |
| |
|
| Platform ID |
GPL13328 |
| Series (2) |
| GSE36726 |
miRNA Profiling in mucosal biopsies of Eosinophilic Esophagitis patients pre and post glucocorticoid steroid treatment |
| GSE36727 |
MicroRNA Profiling in Mucosal Biopsies of Eosinophilic Esophagitis Patients Pre and Post Glucocorticoid Steroid Treatment and Relationship with mRNA Target Expression |
|
