strain: Sprague-Dawley gender: male developmental stage: adult tissue: Third Ventricle Ependyma
Treatment protocol
LCM was performed on an Arcturus® PixCell II® machine. This technique uses a capsule (CapSure® LCM Caps) with a transparent thermosensitive film. Under microscopy, a low power infrared laser beam (980 nm) is used to cut around the edge of the regions of interest, resulting in the melting and adherence of the film to these regions. The optimal laser characteristics were 7.5 μm diameter, 45 mW strength, and 1.4 ms beam time. Cells were captured from the SCO, SFO, PG, and third ventricle ependyma. The ventricular wall cells captured were taken from a region at a distance from the SCO or SFO to avoid contamination and photographs were taken before and after sampling. Only the regions of interest remained on the film when the capsule was removed. The microdissected tissues were released from the film by centrifugation in 20 μl of lysis buffer solution (Qiagen®) and frozen at - 80°C.
Extracted molecule
total RNA
Extraction protocol
The isolated cells in 20 μl of lysis buffer were mixed with 50 μl of lysis buffer and homogenized and RNA extraction performed using a Qiagen RNeasy Micro® kit (Qiagen France SAS, Courtaboeuf, France) following the manufacturer’s instructions. The quality of the total RNA was evaluated on picochips using an Agilent 2100 bioanalyser (RNA 6000 Pico Lab Chip, Agilent Technologies, Massy, France).
Label
CY5
Label protocol
Total RNA (3 ng) was amplified and biotin-labelled by two round of in vitro transcription using a MessageAmp aRNA kit (Austin, Texas, USA) following the manufacturer’s protocol. Before amplification, spikes of different concentrations of synthetic mRNA were added to all samples and were used to determine the quality of all the process. aRNA yield was measured with a UV spectrophotometer and the quality verified on nanoships using Agilent 2100 Bioanalyzer.
Hybridization protocol
Transcriptomic approach was performed on amplified RNA (aRNA) by the genomic platform facility ProfilXpert (Lyon, France) using oligonucleotide microarrays (CodeLink™ Rat Whole Genome Bioarray, General Electric Health Care). Biotin-labelled aRNA (10 ug) from rat tissue were fragmented using 5ug of fragmentation buffer then mixed with 240 ul of Amersham hybridization solution (GE Healthcare Europe GmbH, Saclay, France) and injected on CodeLink™ Rat Whole Genome Bioarray containing 36,736 rat oligonucleotide geneprobes (GE Healthcare Europe GmbH). The arrays were hybridized overnight at 37°C at 15g in an incubator, then washed in stringent TNT buffer at 46°C for 1 h before performing streptavidin-cy5 (GE Healthcare) detection step.
Scan protocol
The arrays were scanned using a Genepix 4000B scanner (Axon, Union City, CA, USA) and Genepix software, with a laser set at 635 nm, the laser power at 60% and the photomultiplier tube voltage at 60%.
Data processing
The scanned image files were analyzed using CodeLink expression software, version 4.2 (GE Healthcare), which produces both a raw and a normalized hybridization signal for each spot on the array. CodeLink software was used to normalize the raw hybridization signal on each array to the median of the array (median intensity is 1 after normalization) for better cross-array comparison. The threshold of detection was calculated using the normalized signal intensity of the 100 negative control samples in the array: spot with signal intensity below this threshold was referred as ‘absent’. The quality of processing was evaluated by generating scatter plots of positive signal distribution.
Molecular Characterization of Laser Microdissected Circumventricular Organs and Third Ventricle Ependyma in the Adult Rat by Microarray Analysis.
Data table header descriptions
ID_REF
Raw_intensity
The spot intensity, which is the difference between the spot mean and the local background median
VALUE
The normalized intensity value (the raw intensity divided byt the normalization factor)
Quality_flag
The metrics used to indicate the quality of a spot : G – The spot has passed all quality control measure and is defined as good. C, I, L, M and S indicate presence of issues
