FACS-RNA: RNA was isolated from FACS-purified Pitx3(gfp/+) mdDA neurons derived from 3 pooled samples of dissected E14.5 embryonic brain (69.000-83.000 cells per sample, each sample contained multiple embryos). FACS-purified neurons were immediately collected in Trizol LS and RNA was isolated according to the manufacturer's protocol with small adaptations to increase purity. RNA for microarray was column purified using the Arcturus Picopure Kit with DNAse treatment on column. In short, a 1:1 mixture of RNA and Extraction Buffer (XB) was combined with an equal volume of 70% EtOH and loaded on column and spun down. Flowthrough was discarded, columns were washed and 40 ul of DNAse mix (35 ul RDD buffer + 5ul DNAseI stock solution (Qiagen RNAse-free DNAse set #79254)) was applied. After 15' incubation at Room Temperature, columns were washed and RNA was eluted according to the manufacturer's protocol.
Label
Cy5
Label protocol
FJ004-cust_lab: Pre-amplification of the RNA was performed using the standard solutions from the QuickAmp labeling kit, no dye from Agilent following a procedure supplied by Agilent on request. For the generation of cDNA, 1.2 ul of T7 promoter primer solution was added to the 10 ul of RNA (1ng/ul), incubated at 65oC for 10 minutes and put on ice. The following was added to the reaction mix: 4 ul of 5x First Strand Buffer, 2ul of 0.1M DTT, 1ul of 10mM dNTP mix, 1ul of MMLV RT Polymerase and 0.5 ul of Rnase Out to a total volume of 20ul. Samples were incubated for 2 hours at 40oC and inactivation was carried out at 65oC for 15 minutes. For in vitro transcription the following was added to the reaction mix: 12.1ul mQ, 20ul of 4x Transcription Buffer, 6ul of 0.1M DTT, 8ul of dNTP mix, 5.6ul of CTP, 6.4ul of 50% PEG, 0.5 ul of Rnase out, 0.6ul of Inorganic Phosphate and 0.8ul of T7 RNA Polymerase to a total volume of 80ul. Reaction mixture was incubated in a waterbath at 40oC for 4 hours. Samples were purified on a Qiagen Rneasy Micro Spin column according to the supplied protocol, and cRNA was eluted in 10.5ul of water. Random hexamer primers were added to the cRNA to a final concentration of 20mM in a final volume of 11.5ul, incubated at 65oC for 10 minutes and stored on ice, after which cDNA was performed as described above. The second round of in vitro transcription, labeling and hybridizations was performed as described by Roepman et al.
FACS-RNA: RNA was isolated from FACS-purified Pitx3(gfp/+) mdDA neurons derived from 3 pooled samples of dissected E14.5 embryonic brain (69.000-83.000 cells per sample, each sample contained multiple embryos). FACS-purified neurons were immediately collected in Trizol LS and RNA was isolated according to the manufacturer's protocol with small adaptations to increase purity. RNA for microarray was column purified using the Arcturus Picopure Kit with DNAse treatment on column. In short, a 1:1 mixture of RNA and Extraction Buffer (XB) was combined with an equal volume of 70% EtOH and loaded on column and spun down. Flowthrough was discarded, columns were washed and 40 ul of DNAse mix (35 ul RDD buffer + 5ul DNAseI stock solution (Qiagen RNAse-free DNAse set #79254)) was applied. After 15' incubation at Room Temperature, columns were washed and RNA was eluted according to the manufacturer's protocol.
Label
Cy3
Label protocol
FJ004-cust_lab: Pre-amplification of the RNA was performed using the standard solutions from the QuickAmp labeling kit, no dye from Agilent following a procedure supplied by Agilent on request. For the generation of cDNA, 1.2 ul of T7 promoter primer solution was added to the 10 ul of RNA (1ng/ul), incubated at 65oC for 10 minutes and put on ice. The following was added to the reaction mix: 4 ul of 5x First Strand Buffer, 2ul of 0.1M DTT, 1ul of 10mM dNTP mix, 1ul of MMLV RT Polymerase and 0.5 ul of Rnase Out to a total volume of 20ul. Samples were incubated for 2 hours at 40oC and inactivation was carried out at 65oC for 15 minutes. For in vitro transcription the following was added to the reaction mix: 12.1ul mQ, 20ul of 4x Transcription Buffer, 6ul of 0.1M DTT, 8ul of dNTP mix, 5.6ul of CTP, 6.4ul of 50% PEG, 0.5 ul of Rnase out, 0.6ul of Inorganic Phosphate and 0.8ul of T7 RNA Polymerase to a total volume of 80ul. Reaction mixture was incubated in a waterbath at 40oC for 4 hours. Samples were purified on a Qiagen Rneasy Micro Spin column according to the supplied protocol, and cRNA was eluted in 10.5ul of water. Random hexamer primers were added to the cRNA to a final concentration of 20mM in a final volume of 11.5ul, incubated at 65oC for 10 minutes and stored on ice, after which cDNA was performed as described above. The second round of in vitro transcription, labeling and hybridizations was performed as described by Roepman et al.
Hybridization protocol
Tecan HS4800 hybridization * 60 ul labeled sample is combined with 60 ul 2x-hybmix, containing 50% formamide, 10xSSC, 0.2% SDS, 200 ug/ml herring sperm DNA * Hybridizations of spotted oligo-arrays (Codelink glass) or Agilent microarrays are performed on a HS4800Pro Hybstation (Tecan) * Priming: 5xSSC, 0.1%SDS * Probe injection: pre-hyb, 5xSSC, 25% formamide, 0.1%SDS, 1%BSA, Volume 110ul * Hybridization: 45 min at 42C. * Wash 2x: milliQ * Wash: 5xSSC, 0.1%SDS * Probe injection: sample. Volume 110ul (Agilent 4packs: 55ul) * Hybridization: 16 hours at 42C. * Wash 2x: 1xSSC, 0.2%SDS at 23C * Wash 2x: 0.1xSSC, 0.2%SDS at 23C * Wash 2x: 0.1xSSC at 23C * Drying: blow with nitrogen for 3min at 30C
Scan protocol
scanning of slides using Agilent G256BA Features were extracted using Imagene software from Biodiscovery.
Data processing
genes, no bg corr - Normalization was done using LOESS as described in (Yang et al. 2002), for all gene probes using no background substraction and a span of 0.4. Probes flagged as absent, or with a (nearly) saturated signal (i.e., > 2^15) in either channel were not considered for the estimation of the LOESS curve.
