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| Status |
Public on Jun 28, 2009 |
| Title |
6-week old mouse cornea sample 3 |
| Sample type |
RNA |
| |
|
| Source name |
wild type C57/BL mouse cornea
|
| Organism |
Mus musculus |
| Characteristics |
tissue: cornea
age: 6-week
strain: C57BL/6
|
| Treatment protocol |
Corneas were dissected from C57BL/6 mice sacrificed at PN9 and 6 weeks (adult) after birth. Total RNAs were isolated from ten 6-week and sixteen PN9 whole corneas. Cornea dissection and RNA extraction for both the 6-week and PN9 corneas were repeated four times to collect altogether eight RNA samples (four adult cornea RNAs and four PN9 cornea RNAs).
|
| Growth protocol |
wild type C57/BL mice at the age of postnatal day 9 (PN9) and 6 weeks after birth were purchased from Charles River.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Trizol according to the manufacturer's protocol (Invitrogen). 5 µg total RNA per sample was size fractionated using a YM-100 Microcon centrifugal filter (Millipore) to isolate small RNAs (<300 nt)
|
| Label |
Cy5
|
| Label protocol |
the small RNAs (< 300 nt) isolated were 3’-extended with a poly(A) tail using poly(A) polymerase. An oligonucleotide tag was then ligated to the poly(A) tail and labeled with Cy5 for later fluorescent detection.
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| |
|
| Hybridization protocol |
The hybridization melting temperatures were balanced by chemical modifications of the detection probes. Hybridization used 100 L 6xSSPE buffer (0.90 M NaCl, 60 mM Na2HPO4, 6 mM EDTA, pH 6.8) containing 25% formamide at 34 °C.
|
| Scan protocol |
Fluorescence images were collected using a laser scanner (GenePix 4000B, Molecular Device) and digitized using Array-Pro image analysis software (Media Cybernetics).
|
| Description |
568 miRNAs were analysed across 4 biological replicates of the mature (6-week-old) mouse cornea
|
| Data processing |
The data process includes background subtraction, Cy5 channel normalization. Background is determined using a regression-based background mapping method. The regression is performed on 5% to 25% of the lowest intensity data points excluding blank spots. Raw data matrix is then subtracted by the background matrix. Normalization is carried out using a cyclic LOWESS (Locally-weighted Regression) method to remove system related variations, such as sample amount variations and signal gain differences of scanners.
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| |
|
| Submission date |
Jun 02, 2009 |
| Last update date |
Jun 02, 2009 |
| Contact name |
Yan Li |
| E-mail(s) |
liyan2@mail.nih.gov
|
| Phone |
3014962764
|
| Organization name |
NIH\NEI
|
| Lab |
LMDB
|
| Street address |
5625 Fishers Lane, Room 1S-02
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL8605 |
| Series (1) |
| GSE16396 |
Differential microRNA expressions during development of mouse cornea |
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