Affymetrix GeneChip Mouse Genome 430A 2.0 [Mouse430A_2]

rawData

scan

processedDataMatrix

Analysis of expression of periglomerular cells from laser microdissection (LMD) samples

cell type

Transcription profiling of mouse periglomerular cells from laser microdissection (LMD) samples

Analysis of expression of periglomerular cells from laser microdissection (LMD) samples


Affymetrix:Protocol:ExpressionStat

Title: Affymetrix CHP Analysis (ExpressionStat). Description:

Affymetrix:Protocol:Hybridization-Unknown

Affymetrix:Protocol:MAS4:ExpressionCall

Title: Affymetrix CHP Analysis (ExpressionCall). Description:

P-AFFY-2

Labeling of samples was carried out according to the manufacturer’s standard protocol

P-AFFY-6

Title: Affymetrix CEL analysis. Description:

P-MEXP-88402

Brain fixation for Laser Microdissection (LMD)Transgenic 5HT3A-EGFP mice (P15) received an intraperitoneal injection of anesthesia (ketanest 18 mg/ml, xylasin 0.24%, final concentration) and were transcardially perfused by 1xPBS for 20 sec (8 ml/min), 0.5% or 2% paraformaldehyde (PFA) for 10 min (8 ml/min) and then by 20% sucrose for 7 min (8 ml/min). After perfusion, the brain was rapidly removed from the skull and frozen on dry ice. Brains were stored at -80C.Preparation of sections for LMDFrozen brains were embedded in Tissue Freezing Medium (Leica Instruments, Germany) at -20C, and 6-8 µm-thick sagittal brain sections were made using the vibratome Microm HM500 (MICROM International, Germany). Sections were mounted on membrane polyester slides (Leica Microsystems, Germany), briefly thawed and dehydrated by sequential incubation in 50% ethanol for 20 sec and n-butanol:ethanol (25:1) for 90 sec, followed by 60 sec of xylene substitution (Sigma-Aldrich, Germany) clearing, to which 1/25 volume of n-butanol was added. Sections were dried for 5 min and used for LMD.Laser MicrodissectionLMD was performed on a Leica LMD6000 B microscope (Leica Microsystems, Germany). Approximately 3,000-5,000 cells were dissected from 50-70 sagittal brain sections of 6-8 µm from one transgenic 5HT3A-EGFP mouse within 4-5 hr. Cells were dissected into dry 0.2 ml tube caps coated with silicon (Leica Microsystems, Germany) at the power 55-57 and speed 3 using a 40x objective or at power 53-54 and speed 3 using a 63x objective. To facilitate collection of such a high cell number, ensembles of adjacent fluorescent cells were often co-dissected during the same laser movement. Three mice were used to assess reproducibility.RNA isolationDirectly following microdissection, the collected 3,000-5,000 cells were lysed in 100 µl of lysis solution [10 mM Tris-HCl (pH 7.9), 50 mM EDTA (pH 7.9), 0.2 M NaCl, 2.2% SDS, 0.5 µg/µl AntiRNase (Ambion, USA) and 1000 µg/ml proteinase K (Ambion, USA)] at 55C for 3h with vigorous shaking. The volume was adjusted to 600 µl by water followed by adding an equal volume of phenol, pH 4.2. The solution was vigorously mixed during 2 min, left for 5 min on ice and centrifuged at 14,000 g for 10 min at 4C. The aqueous phase was aspirated into a fresh tube and subjected to equal volume of phenol:chloroform (1:1) treatment. The mixture was vortexed during 2 min, left for 5 min and centrifuged at 14,000 g for 10 min at 4C. Again, the aqueous phase was transferred into a fresh tube and mixed with an equal volume of isopropanol and 20 µg of glycogen (Ambion, USA). The mixture was incubated at -20C for 30 min and centrifuged at 14,000 g for 10 min at 4C. The pellet was washed with 600 µl of cold 70% ethanol, air-dried and dissolved in 26 µl of water and 3 µl of 10x DNase buffer (Ambion, USA). To eliminate any remaining DNA, the mixture was treated by 1 U of DNase I (Ambion, USA) for 15 min at 37C and purified by use of RNeasy MinElute Cleanup Kit (QIAGEN, Germany) according to manufacturer's recommendations, except for using RPE solution (included in the Cleanup Kit) instead of 80% ethanol in the second washing step and performing the elution step twice, each with 15 µl of water. Obtained RNA (typically 6-9 ng) was concentrated by Eppendorf Concentrator 5301 (Eppendorf, Germany) and analyzed by Bioanalyzer 2100 (Agilent, USA).RNA amplificationTotal RNA (2-3 ng) was amplified using the MessageAmp II aRNA Amplification Kit (Ambion, USA) according to manufacturer's recommendations. During the T7 in vitro transcription step, the mixture was incubated at 37C for 14-16h. After each amplification round, the RNA was analyzed by Bioanalyzer 2100 (Agilent, USA). We typically obtained 100-200 ng and 50-100 µg of amplified RNA after the first and second amplification round, respectively. Amplifications were done from three periglomerular cell RNA samples obtained from three 5HT3A-EGFP mice.

Transcription profiling of mouse periglomerular cells from laser microdissection (LMD) samples

Submitter Supplied Information

Samples