|
| Status |
Public on Jan 07, 2019 |
| Title |
TA-Nuc-3 |
| Sample type |
RNA |
| |
|
| Source name |
TA myofiber pool, nuclear fraction, biological replicate 3
|
| Organism |
Mus musculus |
| Characteristics |
strain: CD1
gender: female
tissue: TA muscle
age: 3 months
|
| Treatment protocol |
Muscles were digested in type I collagenase (10mg/ml in DMEM) and single myofibers were dissociated. Intact (non contracted) single myofibers were picked under a sterereomicroscope and washed in PBS. Pools of 5-10 myofibers were lysed in RLN Buffer (Water, 50 mM TrisHCl pH 8.0, 140 mM NaCl, 1.5 mM MgCl2, 0.5% v/v Triton-X100, 0.36 units/µl RNase OUT) by repeatedly passing the solution through a 0.2 μm needle. Nuclei were then precipitated in a microcentrifuge for 5 min at 600 g, 4°C. Surnatant volume was reduced by 50% in a Savant SpeedVac. Total RNA was extracted from each fraction.
|
| Growth protocol |
CD1 female mice were housed in a normal environment provided with food and water and were killed by rapid cervical dislocation to minimize suffering.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
1 ml of TRIzol Reagent (Thermo Fisher Scientific) was used to extract RNA from each purified fraction of nuclei or cytoplasm. 300 μl of aqueous phase were additioned with 300 μl of isopropanol, samples were stored o/n at -20°C and then centrifuged for 20 min at 12,000 x g, 4°C to precipitate RNA. Pellet was washed twice with 70% EtOH and resuspended in 20 μl of DNase/RNase free water.
|
| Label |
Cy3
|
| Label protocol |
Fluorescent cRNA to hybridize onto microarray was produced by Low Input Quick Amp Labeling Kit (Agilent) according to manufacturer instructions.
|
| |
|
| Hybridization protocol |
Labeled cDNA were mixed with 5 µl of 10X Blocking Agent (Agilent Technologies) and water to a final volume of 25 µl. Samples were denatured at 95°C for 2 min. and added to 25 µl of 2X GEx Hybridization Buffer HI-RPM (Agilent Technologies). 40 µl mix was dispensed onto one array of the SurePrint G3 Mouse Gene Expression 8x60K microarray. Slides were loaded into the Agilent SureHyb chambers and hybridization was performed in a hybridization oven at 65°C for 17 hours with 10 rpm rotation. Finally, slides were washed using Wash Buffer Kit (Agilent Technologies) and dried at room temperature.
|
| Scan protocol |
Microarrays were scanned using the Agilent Technologies Scanner G2505C US22502723 (ChipScan software version A.8.5.1), scan region 61 x 21.6 mm, scan resolution 3 micron single pass, Green PTM 100%, no extended dynamic range (NO XDR).
|
| Description |
RNA extraction from the nuclear fraction of a pool containing 5-10 TA myofibers from mouse identified as 3 (Nuclei-TA-3)
|
| Data processing |
Data was extracted from the scanned image using the Agilent Feature Extraction Software version 10.7.3.1 (protocol GE1_107_Sep09). Microarray probes were re-annotated according to their ability to match to transcripts in Ensembl 74 and probes identifying coding genes and non-coding genes were treated separately because their different expression. Non-coding RNAs are lesser expressed than coding RNAs and therefore corresponding signal intensity is smaller. Quantile inter-arrays normalization was performed in R statistic ambient considering only the gProcessedSignal column for probes identifying non-coding RNAs. Normalized values only for non-coding RNAs are indicated. Probes with gIsPosAndSignif value of zero were disregarded and labeled as “NA”. Mean values were calculated for multiple probes with the same ProbeName (Agilent Probe ID).
|
| |
|
| Submission date |
Apr 05, 2018 |
| Last update date |
Jan 07, 2019 |
| Contact name |
Gerolamo Lanfranchi |
| E-mail(s) |
stefano.cagnin@unipd.it
|
| Phone |
+39-0498276219
|
| Organization name |
University of Padova
|
| Department |
CRIBI - Biotechnology Center and Biology Department
|
| Lab |
Functional Genomics Lab
|
| Street address |
Via U. Bassi, 58/B
|
| City |
Padova |
| ZIP/Postal code |
35131 |
| Country |
Italy |
| |
|
| Platform ID |
GPL24842 |
| Series (1) |
| GSE112768 |
Subcellular localization analysis of long non-non coding RNAs (lncRNAs) in skeletal muscle myofibers |
|
