yeast RNA isolation for robotic amplification v1.0
Label
Cy5
Label protocol
robot amplification and labeling v1.0: All amplification and labeling procedures are performed in 96 wells plates (4titude, Bioke) on a customized Sciclone ALH 3000 Workstation (Caliper LifeSciences), supplemented with a PCR PTC-200 (Bio-Rad Laboratories), SpectraMax 190 spectrofotometer (Molecular Devices), and a magnetic bead-locator (Beckman). mRNA amplification protocol description: * all RNAs are diluted to 0.6 ug/ul and 5 ul is put in a 96-wells plate (Abgene). All subsequent steps are performed by the robot script. * mix1 containing 100 ng T7 Mlu VN primer (custom mix) and EC-control RNA per 5 ul is added and mixed in each well. * plate is incubated @ 70C for 10 mins, cooled to 48C. * mix2 containing 4ul 5x 1st strand buffer, 2 ul 0.1M DTT (Invitrogen), 1ul RNAse Inhibitor (Boehringer), 1ul 20mM dNTPs (GE Healthcare), 1ul linear acrylamide, and 1ul SuperScriptIII (Invitrogen) per sample is prewarmed to 48C; 10 ul per sample is added and mixed in each well. * plate is incubated @ 48C for 2 hours and cooled to room temperature. * 106 ul water and subsequently mix3 containing 15ul second strand buffer, 3ul 20mM dNTPs (GE Healthcare), 1 ul T4 DNA ligase, 4 ul E.coli DNA polymerase I and 1 ul RNAseH (Promega) is added and mixed in each well. * plate is incubated @ 16C for 2 hours, @65C for 10 mins. * ds cDNA product is purified and concentrated with RNAClean (Agencourt, GC biotech) according to manufacturers protocol, to an end volume of 25 ul . * 8 ul cDNA is put in a 96 wells plate and mixed with 12 ul IVT mix containing 2 ul 10x rxn-buffer, 2 ul each ATP, CTP, GTP, 0.6 ul UTP, 2 ul T7 enzyme mix (MegaScript kit, Ambion, Applied Biosystems), and 2.1 ul 50 mM 5-(3-aminoallyl)-UTP (Ambion, Applied Biosystems). * Plate is incubated @37C for 4 hours. * cRNA product is purified with RNAClean (Agencourt, Beckman) according to manufacturers protocol. * Concentration is measured (SpectraMax 190) and adjusted to 600 ng/ul by the robot. * Resulting cRNA plates are sampled for Bioanalyzer QC, snapfrozen and stored at -80C. Labeling protocol description: * NHS-ester Cy3 or Cy5 dye (Amersham PA 23001 and 25001): entire tube is resuspended in 100 ul DMSO (Merck 8.02912.10). All subsequent steps are performed by the robot script : * 8 ul of each cRNA (0.6 ug/ul) is put in a 96-wells plate (Abgene), the accompanying reference sample is put in the next column (final step combines cy3 and cy5 labeled material ). * 3 ul 0.5 M NaBicarbonate buffer, pH 9 is added and mixed to all wells. * 3 ul cy-dye solution is added and mixed to the appropriate wells, plate is incubated at 18C for 1 hour. * 4.5 ul 5M hydroxylamine is added and mixed, incubated at 18o C for 15 minutes. * Labeled cRNA product is purified with RNA Clean (Agencourt, GC biotech) according to manufacturers protocol. * RNA concentration and labeling incorporation are measured (SpectraMax 190). * 2.5 ug of each labeled sample and reference cRNA are pooled and subjected to fragmentation according to protocol (Ambion, Applied Biosystems), 15 min 70C. * Samples are stored at -20C until hybridization.
Yeast growth for expression profiling in Tecan platereader v1.0
Extracted molecule
polyA RNA
Extraction protocol
yeast HTP RNA isolation for robot v2.0
Label
Cy3
Label protocol
robot amplification and labeling v1.0: All amplification and labeling procedures are performed in 96 wells plates (4titude, Bioke) on a customized Sciclone ALH 3000 Workstation (Caliper LifeSciences), supplemented with a PCR PTC-200 (Bio-Rad Laboratories), SpectraMax 190 spectrofotometer (Molecular Devices), and a magnetic bead-locator (Beckman). mRNA amplification protocol description: * all RNAs are diluted to 0.6 ug/ul and 5 ul is put in a 96-wells plate (Abgene). All subsequent steps are performed by the robot script. * mix1 containing 100 ng T7 Mlu VN primer (custom mix) and EC-control RNA per 5 ul is added and mixed in each well. * plate is incubated @ 70C for 10 mins, cooled to 48C. * mix2 containing 4ul 5x 1st strand buffer, 2 ul 0.1M DTT (Invitrogen), 1ul RNAse Inhibitor (Boehringer), 1ul 20mM dNTPs (GE Healthcare), 1ul linear acrylamide, and 1ul SuperScriptIII (Invitrogen) per sample is prewarmed to 48C; 10 ul per sample is added and mixed in each well. * plate is incubated @ 48C for 2 hours and cooled to room temperature. * 106 ul water and subsequently mix3 containing 15ul second strand buffer, 3ul 20mM dNTPs (GE Healthcare), 1 ul T4 DNA ligase, 4 ul E.coli DNA polymerase I and 1 ul RNAseH (Promega) is added and mixed in each well. * plate is incubated @ 16C for 2 hours, @65C for 10 mins. * ds cDNA product is purified and concentrated with RNAClean (Agencourt, GC biotech) according to manufacturers protocol, to an end volume of 25 ul . * 8 ul cDNA is put in a 96 wells plate and mixed with 12 ul IVT mix containing 2 ul 10x rxn-buffer, 2 ul each ATP, CTP, GTP, 0.6 ul UTP, 2 ul T7 enzyme mix (MegaScript kit, Ambion, Applied Biosystems), and 2.1 ul 50 mM 5-(3-aminoallyl)-UTP (Ambion, Applied Biosystems). * Plate is incubated @37C for 4 hours. * cRNA product is purified with RNAClean (Agencourt, Beckman) according to manufacturers protocol. * Concentration is measured (SpectraMax 190) and adjusted to 600 ng/ul by the robot. * Resulting cRNA plates are sampled for Bioanalyzer QC, snapfrozen and stored at -80C. Labeling protocol description: * NHS-ester Cy3 or Cy5 dye (Amersham PA 23001 and 25001): entire tube is resuspended in 100 ul DMSO (Merck 8.02912.10). All subsequent steps are performed by the robot script : * 8 ul of each cRNA (0.6 ug/ul) is put in a 96-wells plate (Abgene), the accompanying reference sample is put in the next column (final step combines cy3 and cy5 labeled material ). * 3 ul 0.5 M NaBicarbonate buffer, pH 9 is added and mixed to all wells. * 3 ul cy-dye solution is added and mixed to the appropriate wells, plate is incubated at 18C for 1 hour. * 4.5 ul 5M hydroxylamine is added and mixed, incubated at 18o C for 15 minutes. * Labeled cRNA product is purified with RNA Clean (Agencourt, GC biotech) according to manufacturers protocol. * RNA concentration and labeling incorporation are measured (SpectraMax 190). * 2.5 ug of each labeled sample and reference cRNA are pooled and subjected to fragmentation according to protocol (Ambion, Applied Biosystems), 15 min 70C. * Samples are stored at -20C until hybridization.
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: Scanning of slides using the Agilent G2565BA scanner. Imagene feature extraction: Features were extracted using Imagene software from Biodiscovery.
Data processing
genes, no bg corr, dye corr: Normalization was done using print-tip LOESS as described in (Yang et al. 2002), by estimating the LOESS curve 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. Signals were corrected for dye bias using intrinsic gene-specific dye biases estimated from a set of wild-type wild-type hybridizations, as described in Margaritis, Lijnzaad et al., Mol. Sys. Biol. 2009. limma: A software package for the analysis of gene expression microarray data, especially the use of linear models for analysing designed experiments and the assessment of differential expression. Author(s): Gordon Smyth. The limma R package version 2.12.0 is used. P-values are Benjamini-Hochberg FDR corrected.
