|
| Status |
Public on Jul 10, 2018 |
| Title |
sham-24h-5 (exosomal RNA) |
| Sample type |
RNA |
| |
|
| Source name |
plasma exosome
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6J
genotype: WT
disease state: control
rna source: plasma exosome
gender: Male
time: 24 hours post-surgery
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Exosome was purfied from plasam via ultracentrifuge. RNA was extracted for plasma exosome using miRNeasy Micro Kit (Qiagen)
|
| Label |
Fireplex
|
| Label protocol |
Oligonucleotide adapters are ligated to each end of the hybridized miRNA to serve as PCR priming sites. Labelled miRNAs are dehybridized from particles and undergo one-step RT-PCR with a biotinlyated primer.
|
| |
|
| Hybridization protocol |
For each sample, FirePlex Particles (35 μl) are added to a well of 96-well flitler plate and fitlered. Three additional wells for no-sample controls are also prepared. Next, 25 μl of Hybridization Buffer is added to each well followed by 25 μl sample. The plate is incubated at 37oC for 60 minutes while shaking at 750 rpm
|
| Scan protocol |
Particles are scanned on an EMD Milipore Guava 8HT or 6HT flow cytometter
|
| Description |
R-S-105
|
| Data processing |
The flow cytometer output was analyzed with the FirePlexTM Analysis Wrokbench software, avaialble on the Abcam website at www.abcam.com/FireflyAnalysisSoftware, and R.
[normalization method applied to the signal intensity data]:
A geNorm-like algorithm is applied to the data in order to select the three most appropriate miRNAs for normalization in the experiment. To improve the likelihood of identifying three appropriate normalization candidates, several miRNA targets identified by others [Chen et al] and by Abcam in an independent analysis are automatically included in the fixed panels. The miRNAs selected for normalization of the current samples are: hsa-let-7i-5p, hsa-mir-20a-5p, hsa-miR-29a-3p.
Chen X., Liang H., Guan D., Wang C., Hu X., et al. (2013) A Combination of Let-7d, Let-7g and Let-7i Serves as a Stable Reference for Normalization of Serum microRNAs. PLoS ONE 8(11): e79652.
|
| |
|
| Submission date |
Jul 09, 2018 |
| Last update date |
Jul 10, 2018 |
| Contact name |
Lin Zou |
| E-mail(s) |
lzou@som.umaryland.edu
|
| Organization name |
University of Maryland School Of Medicine
|
| Department |
Anesthesiology
|
| Street address |
20 Penn Street, HSF II-GS003C
|
| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21201 |
| Country |
USA |
| |
|
| Platform ID |
GPL25310 |
| Series (1) |
| GSE116829 |
Plasma exosomal miRNAs microarray in polymicrobial sepsis |
|
