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Online Resource 2 Elstner et al.
A) LMD Experimental Design and Methods
Figure: Workflow of LMD study
1) Cryosectioning: Frozen midbrains were equilibrated in the cryotome to -20°C for 60 min. Midbrains were sectioned at 20µm thickness and transferred to Leica PEN-slides (pre-treated in UV crosslinker for 30 min). Slides were kept in the cryostat until sections were re-frozen. Five sections were transferred to slide mailers at a time, sealed air-tight and transferred to -80°C until further processing
2) Staining: Toluidine blue was used for LMD (Vega CJ. Laser microdissection sample preparation for RNA analyses. Methods Mol Biol. 2008;414:241-52). Before LMD, slides were individually transferred from -80°C into ice-chilled 100% Ethanol (1 min) and sections stained in a toluidine blue solution (1% in RNAse free H2O) for 30 seconds. Staining was followed by an alcohol dehydration series (50%, 75%, 95%, 2x 100%). Slides were air-dried for 10 minutes and immediately used for LMD.
3) LMD: Midbrain DA neurons were identified by toluidine stain and neuromelanin pigment. Only neurons with a clear visible soma and pigment were dissected. 100 neurons were dissected following the outlines of the toluidine stained soma and successful capture was visually confirmed. After collection, 50µl Arcturus PicoPure® RNA buffer was added to the capture vessel and mixed thoroughly, spun down at maximum speed for 10 minutes and transferred to -80°C.
a) Midbrain block in cryostat
b) Overview of toluidine blue stained midbrain section
c) The same section after LMD
d) Toluidine blue stained midbrain neurons and clear visible neuromelanin
e) Neuron dissected; LMD of a 2nd neuron was stopped in the process (arrow)
f) Visual confirmation of dissected neurons in the capture vessel
4) RNA was extracted on the day of IVT, using the Arcturus PicoPure® Kit (Applied Biosystems, Carlsbad, CA, USA) following the manufacturers protocol. RNA was eluted in 10µl RNAse-free water and used directly for IVT without any prior storage.
5) All RNA was used for IVT. For the first round, the Ambion MessageAmpII® was used following the manufacturers recommendations (Applied Biosystems, Carlsbad, CA, USA). After the first round, aRNA was eluted in 100µl RNAse-free H2O and the volume was reduced to 10µl in a vacuum centrifuge. The second round IVT was performed using the Illumina® TotalPrep™ RNA Amplification Kit (Applied Biosystems, Carlsbad, CA, USA). The second round of IVT yielded >3µg cRNA with an average length of 800 bp and was used for hybridization on Illumina® WG6v1 expression chips (Illumina®, San Diego, CA, USA).
B) Mircoarray Experimental Design (MIAME)
Type of Experiments
Comparison of gene expression profiles of midbrain dopaminergic neurons collected from Parkinson’s disease patients, age matched controls and middle-aged controls.
Experimental factors
I. Single cells vs. substantia nigra homogenate.
II. Age, Disease
Number of hybridizations performed
48 samples LMD samples (100 DA neurons from human substantia nigra pars compacta). 2 samples from dissected substantia nigra of human midbrain slices.
Hybridization design
Samples were randomly distributed over 9 microarray chips.
Quality control steps taken
I. Tissue factors: pH, RIN
II: Duplicates, detected genes, cluster analysis
Number of replicates
11 PD cases (PD), 11 age-matched controls (old controls; OC) and 8 young controls (YC) met the screening criteria of pH≥6.3 and RNA integrity numbers (RIN) >6 (determined on an Agilent 2100 bioanalyzer) and were eventually suitable for RNA isolation, in vitro transcription (IVT) and microarray hybridization.
URL and any supplemental websites or database accession numbers
All primary files will be placed on a supplemental website prior to publication
Chip positions and sample identifiers
Chip Identifier / Position / Sample / Used / Comments1401771035 / A / YC5 / + / Replicates
B / YC5 / +
C / YC5 / +
D / YC5 / +
E / YC5 / (+) / Bulk tissue homogenat, biological replicates
F / YC5 / (+)
1401771266 / A / OC / - / Cluster outlier
B / PD1 / +
C / PD8 / +
D / These samples do not pertain to this study
E
F
1401771267 / A / YC / - / Replicates, cluster outlier
B / YC / -
C / OC8 / +
D / PD7 / +
E / OC5 / +
F / PD4 / +
1401771274 / A / YC7 / + / Replicates
B / YC7 / +
C / YC2 / + / Replicates
D / YC2 / +
E / YC3 / + / Replicates
F / YC3 / +
1401771277 / A / YC1 / + / Replicates
B / YC1 / +
C / YC6 / + / Replicates
D / YC6 / +
E / YC4 / +
F / YC4 / - / Cluster outlier
1401771286 / A / PD6 / + / Replicates
B / PD6 / +
C / PD2 / - / Cluster outlier
D / PD2 / +
E / PD / - / Cluster outlier
F / OC9 / +
1401771287 / A / OC1 / + / Replicates
B / OC1 / +
C / OC2 / - / Cluster outlier
D / OC2 / +
E / PD / - / Replicates, cluster outlier
F / PD / -
1401771288 / A / OC4 / + / Replicates
B / OC4 / +
C / OC / - / Replicates, cluster outlier
D / OC / -
E / PD5 / + / Replicates
F / PD5 / +
1401771295 / A / OC7 / +
B / X / - / Outlier, revised diagnosis
C / OC3 / +
D / OC8 / +
E / PD / - / Cluster outlier
F / PD3 / +
Samples, Preparation, and Labeling
Biological samples
Frozen human brain tissue was obtained from individuals who had a history of PD and from age matched and younger control individuals without neurological disease. The cases were clinically well documented and neuropathologically confirmed. Inclusion criteria comprised the neuropathological diagnosis of Lewy body disease (LBD) with typical pathological features, including moderate to severe neuronal loss and gliosis. Procedures were approved by the Local Research Ethics Committee.
RNA Isolation and Processing
See materials and methods.
Hybridization Procedures and Parameters
Standard hybridization protocol was performed according to the manufacturer’s recommendations.
Measurement Data and Specifications
The Illumina® BeadArray Reader was used to scan the surface of beadarrays. The digital images from the intensity signals were stored as .tiff files and the corresponding signal intensity values were decoded using the Decode map files provided with each beadarray and converted into the Illumina® propriety format .idat files. After scanned microarray images of Sentrix® BeadChips collected from the Illumina® BeadArray Reader, image data files (.idat) were directly downloaded into BeadStudio for data visualization and analysis.
Array Design
Platform Type
Sentrix Human WG-6 V1 Expression BeadChip. Synthesized 50-mer randomly assembled Oligonucleotide Array. Silica beads deposited on a glass surface
Availability of the Array
Beadarrays annotation files can be downloaded at:
http://www.illumina.com/support/annotation_files.ilmn