6 protocols
AccessionNameType
P-MTAB-27055
bioassay_data_transformation
The results for each replica (median intensity for each channel) were normalized and statistically analyzed using the LIMMA software package (Smyth, 2004), a part of Bioconductor (an R-language project; R Development Core Team, 2004). Background subtraction was performed using “normexp”, a method implemented in LIMMA designed to yield positive corrected intensities (i.e., to avoid negative intensity values): a convolution of normal and exponential distributions was fitted to the foreground intensities, using the background intensities as covariate. This results in a smooth monotonic transformation of the background subtracted intensities such that all the corrected intensities are positive. Differential expression was calculated using linear models and empirical Bayes moderated t-statistics (Smyth and Speed, 2003; Smyth, 2004). The resulting log-ratios were normalized for each array through loess (Smyth and Speed, 2003) and expression values were scaled to achieve consistency among arrays by using the “quantile” method. Each probe was tested for changes in expression over replicates by using moderated t-statistics (Smyth, 2004). The p-values were adjusted for multiple testing as described (Benjamini and Hochberg, 1995) to control the false discovery rate.



M=log2(Ratio)

SE= Standard Error of M

p.value=raw p.value

adj. p.value (FDR)=adjusted p.value



Ref: Benjamini, Y., and Hochberg, Y. (1995) J R Stat Soc B 57: 289-300. R Development Core Team (2004) R: A lenguage and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing. Smyth, G.K., and Speed, T. (2003) Methods 31: 265-273. Smyth, G.K. (2004) Stat Appl Genet Mol Biol 3: nº1, article 3.
P-MTAB-27054
hybridization
Previous to the hybridization process, the microarray was blocked by immersion into a 50 ml Falcon tube containing 5x SSC, 0.1% (w/vol) SDS, 1% (w/vol) bovine serum albumin, preheated to 42ºC. After 45 min at 42ºC, the microarray slide was washed by a brief immersion into a Falcon tube containing H2O at RT, followed by another immersion in isopropanol. The slide was then allowed to dry.



Equal amounts of Cy3- or Cy5-labeled cDNAs (about 300 pmoles each), one of them corresponding to the control and the other one to the problem to be analyzed, were mixed and dried in a Speed-Vac. The sample was dissolved in 35 ?l of a solution containing 50% (vol/vol) deionized formamide, 5x Denhardt’s solution, 6x SSC, 0.5 (w/vol) SDS, 5% (w/vol) dextransulphate, pre-filtered and preheated to 42ºC. After 2 min at 90ºC to denature the cDNA, the solution was applied to the microarray slide and covered with a 24x60 mm cover glass. The slide was introduced into a hybridization chamber and incubated at 42ºC for 18 hours, preserved from light. The microarray was then transferred to a Falcon tube containing 0.5x SSPE (1x SSPE contains 150 mM NaCl, 1 mM EDTA, 11.5 mM NaH2PO4, pH 7.4), 0.5% (w/vol) SDS, preheated to 37ºC. After eliminating the cover glass, the microarray was washed by gentle agitation for 5 min. The slide was transferred to a new tube with 0.5x SSPE, 0.5% (w/vol) SDS at RT, and washed again with gentle shaking for 5 min. Similar washes were performed three times in 0.5x SSPE at RT, and once in 0.1X SSPE.

(Parameters: Chamber type = OTHER: other, Quantity of label target used = 100, Mass unit = Nano gram, time = 18, Tiny time unit = hours, Volume = 35, Volume unit = Micro litre, temperature = 42)
(Parameters: Chamber type = OTHER: other, Quantity of label target used = 100, Mass unit = Nano gram, Tiny time unit = seconds, Volume unit = Nano litre, temperature = 42)
P-MTAB-27053
labeling
Fluorescently labelled cDNA for microarray hybridizations was obtained by using the SuperScript Indirect cDNA Labelling System (Invitrogen), as recommended by the supplier. In brief, 20 ?g of total RNA were transformed to cDNA with Superscript III reverse transcriptase using random hexamers as primers, and including aminoallyl-modified and aminohexyl-modified nucleotides in the reaction mixture. After cDNA purification, the Cy5 fluorescent dye (Amersham Biosciences) was coupled to the amino-modified first-strand cDNA. Labelling efficiency was assessed using a NanoDrop ND1000 spectrophotometer (NanoDrop Technologies).

(Parameters: Amount of nucleic acid labeled = 20, Mass unit = Micro gram, Amplification = none)
(Parameters: Amplification = none, Mass unit = Micro gram)
P-MTAB-27056
nucleic_acid_extraction
To obtain total RNA, four independent cultures were grown for each strain (wt and PP3546 mutant) in LB medium, harvested at mid-exponential phase (A600=0.65) by centrifugation and quick chilling, and the pellets were frozen at –70ºC. RNA was purified from each pellet with RNeasy RNA purification kit (Qiagen), using RNase-Free DNase set for DNase treatment on column following manufacturer's instructions.
(Parameters: Extracted product = total_RNA, Amplification = none)
P-MTAB-27057
image_acquisition
The microarray was allowed to dry and scanned in a Microarray scanner with green and red lasers operating at 543 and 633 nm, respectively, to excite Cy3 and Cy5. Images were taken at 10 ?m resolution and spot intensity was determined using the software package ScanArray Express (PerkinElmer)

(Parameters: Scanning hardware = ScanArray Lite [PerkinElmer], Scanning software = ScanArray Express [PerkinElmer])
(Parameters: Scanning hardware = ScanArray Lite [PerkinElmer], Scanning software = ScanArray Express [PerkinElmer])
P-MTAB-27058
labeling
Fluorescently labelled cDNA for microarray hybridizations was obtained by using the SuperScript Indirect cDNA Labelling System (Invitrogen), as recommended by the supplier. In brief, 20 ?g of total RNA were transformed to cDNA with Superscript III reverse transcriptase using random hexamers as primers, and including aminoallyl-modified and aminohexyl-modified nucleotides in the reaction mixture. After cDNA purification, the Cy3 fluorescent dye (Amersham Biosciences) was coupled to the amino-modified first-strand cDNA. Labelling efficiency was assessed using a NanoDrop ND1000 spectrophotometer (NanoDrop Technologies).

(Parameters: Amount of nucleic acid labeled = 20, Amplification = none, Mass unit = Micro gram)
(Parameters: Amplification = none, Mass unit = Micro gram)