S. cerevisiae (rlm1 mutant strain BY4741) was grown on YEPD. For Zymolyase experiments, yeast cells were grown overnight at 24 °C to an optical density 0.8 - 1 (A600). The culture was refreshed to 0.2 O.D and grown at 24 °C for 2h. Next, culture was divided into two parts. One continues growing under same conditions (non-treated culture) while the other was supplemented with Zymolyase to a final concentration of 0,8U/ml. Cells were collected at 3 hours of growth, frozen at -80 °C and processed for RNA extraction.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from exponentially growing cells (5 x 108) by the “mechanical disruption protocol” using the RNeasy MIDI kit (Qiagen, Hilden, Germany), following the instructions of the manufacturer. RNA concentrations were determined by measuring absorbance at 260 nm. RNA purity and integrity were assessed using RNA Nano Labchips in an Agilent 2100B Bioanalyzer (Agilent Technologies, Palo Alto, CA) following the manufacturer’s instructions.
Label
Cy5
Label protocol
cDNA was synthesized from 25–30 μg of total RNA by reverse transcription, using the CyScribeTM First-Strand cDNA Labeling Kit, incorporating Cy5- dUTP (Amersham Biosciences) into the cDNA corresponding to each sample to be compared. Labeled cDNA was dried in a vacuum trap and used as a hybridization probe after resuspension in 45 μl of hybridization solution (50% Formamide, 6 x SSC, 0.5% SDS, 5 x Denhardt’s, 20 μg of poly(A) (P-9403, Sigma) and salmon sperm (Invitrogen, 100 μg/ml)
Channel 2
Source name
total RNA Saccharomyces cereviase rlm1 mutant, without zymolyase
S. cerevisiae (rlm1 mutant strain BY4741) was grown on YEPD. For Zymolyase experiments, yeast cells were grown overnight at 24 °C to an optical density 0.8 - 1 (A600). The culture was refreshed to 0.2 O.D and grown at 24 °C for 2h. Next, culture was divided into two parts. One continues growing under same conditions (non-treated culture) while the other was supplemented with Zymolyase to a final concentration of 0,8U/ml. Cells were collected at 3 hours of growth, frozen at -80 °C and processed for RNA extraction.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from exponentially growing cells (5 x 108) by the “mechanical disruption protocol” using the RNeasy MIDI kit (Qiagen, Hilden, Germany), following the instructions of the manufacturer. RNA concentrations were determined by measuring absorbance at 260 nm. RNA purity and integrity were assessed using RNA Nano Labchips in an Agilent 2100B Bioanalyzer (Agilent Technologies, Palo Alto, CA) following the manufacturer’s instructions.
Label
Cy3
Label protocol
cDNA was synthesized from 25–30 μg of total RNA by reverse transcription, using the CyScribeTM First-Strand cDNA Labeling Kit, incorporating Cy3-dUTP (Amersham Biosciences) into the cDNA corresponding to each sample to be compared. Labeled cDNA was dried in a vacuum trap and used as a hybridization probe after resuspension in 45 μl of hybridization solution (50% Formamide, 6 x SSC, 0.5% SDS, 5 x Denhardt’s, 20 μg of poly(A) (P-9403, Sigma) and salmon sperm (Invitrogen, 100 μg/ml)
Hybridization protocol
Slides were prehybridized in prehybridization solution (6 x SSC, 0.5% SDS, 1% bovine serum albumin (A-7906, Sigma) for 1 h and were then hybridized overnight in a LucideaTM SlidePro Automated Hybridization Station (Amersham Biosciences). Before scanning, the chips were washed and dried in a same LucideaTM SlidePro Automated Hybridization
Scan protocol
Microarrays were scanned with aGenePix 4000B scanner (Axon Instruments, Union City, CA) at a resolution of 5 μm (PMT values ranging from 550 to 700 and laser power 100%). GenePix Pro 4.0 analysis software (Axon Instruments, Union City, CA) was used to locate spots in the microarray with the appropriate grid and to obtain the two Cy3/Cy5 image TIFF files. All images were further processed using GenePix 4.0 software according to the manufacturer’s instructions.
Description
For each condition tested, comparison of treated and non-treated samples, the total RNA from two different cultures was analyzed, and, in addition, for each sample two different hybridizations were performed, including fluorochrome swapping in order to minimize transcriptional changes due to technical variability. This therefore corresponded to four DNA microarrays analyzed for each condition.
Data processing
Flagged spots and spots with an average intensity minus background below the mean of the background (considering this as the median of the local background for each spot) for all the non-flagged spots in any of the channels (Cy3 or Cy5) were not retained for further analysis. Within this group, the spots showing in one channel a value of intensity minus background higher than 5 times the mean of the background for all spots in the microarray for that channel were recovered. The reproducibility of replicates in each microarray (two spots per ORF) was analyzed by creating a normal distribution log2 (RA _ 1/RB), where RA and RB are the ratios of replicated spots after background subtraction and normalization. Replicates that exceeded the average by more than _3 S.D. were discarded. Data analysis was accomplished using the GeneSight 4.0 software package (BioDiscovery Inc, El Segundo, CA). Locally weighed linear regression (lowess) analysis was performed as a normalization method to remove the intensity-dependent deviation in the log2(ratio) values. Significance analysis of the results was conducted using a Student’s t test (GeneSight). Genes with p values of less than 0.05 were considered to be significantly differentially expressed with respect to the treatment condition.
