Total RNA isolation was performed using the RNeasy Protect Bacteria system from Qiagen.
Sample collection from M9 medium Grow bacteria overnight in LB medium, reinoculate in M9 minimal medium at an initial OD600 of 0.005. After eight hours growth at 34°C in M9 medium, 2 mL of RNA Protect Reagent (Qiagen) was added to 1 ml bacterial cultures (at OD600 of about 0.5-0.8) to stabilize RNA. Centrifuge for 10 min at 4000 g. Decant the supernatant.
Bacterial lysis Add 100 μl 1 mg/ml lysozyme buffer (TE buffer: 10 mM Tris-HCL, 1 mM EDTA pH=8.0 containing 1 mg/ml lysozyme). Vortex for 10 s and incubate at RT for at least 15 min. Add 350 μl Buffer RLT and vortex vigorously. Add 250 μl 100% ethanol, mix by pipetting.
Column purification Transfer 700 μl lysate into RNeasy Mini column place in a 2 ml collection tube and centrifuge for 30 s at highest speed. Add 350 μl Buffer RW1 to the column and centrifuge for 30 s. Discard flow-through and reuse the collection tube. Add 10 μl DNase stock solution to 70 μ l RDD Buffer. Add the DNase I solution into the column and incubate at RT for 15 min. Add 350 μl Buffer RW1 to the column and wait for 5 min, centrifuge for 30 s. Discard the flow-through and collection tubes. Place the RNeasy Mini spin column in a new 2 ml collection tube. Add 500 μl Buffer RPE to the column. Centrifuge for 30 s. Place the column in a new 1.5 ml tube, and centrifuge for 1 min to eliminate any residue ethanol. Place the column in a new collection tube. Add 50 μl RNase free water, centrifuge for 1 min. Store RNA samples at -20°C.
Label
Alexa Fluor 555
Label protocol
cDNA labeling was carried out using the FairPlay III Microarray Labeling Kit from Agilent.
Reverse transcription 10 μ g total RNA was used to generate cDNA. Add 1 μl of 500 ng/ μl random primers into the 10 μg total RNA which is dissolved in 12 μl RNase free water. Incubate at 70°C for 10 min in a PCR machine. Cool samples immediately on ice until ready for use. Combine the following components in a sterile, RNase and DNase free tube: 2 μl 10 × AffinityScript RT Buffer; 1 μl 20 × dNTP mix; 1.5 μl 0.1 M DTT; 0.5 μl 40 U/ul RNase Block. Add the annealed primers and RNA into the mixture. Add 3 μl of AffinityScript RT and incubate at 42°C for 60 min. Add 10 μl of 1 M NaOH and incubate at 70°C for 10 min to hydrolyzed RNA. Cool to room temperature and add 10 μl 1 M HCL to neutralize the solution.
cDNA purification Add 4 μl of 3 M sodium acetate pH 5.5 to the reaction. Add 1 μl of 20 mg/ml glycogen to the reaction. Add 100 μl ice cold 100% ethanol and incubate at -20°C overnight. Spin the reaction at 13.2 rpm for 15 min at 4°C and decant the supernatant (You should see some white pellet at the bottom of the tube). Wash with 200 μl ice cold 70% ethanol and spin 5 min. Decant supernatant carefully and allow for air dry for about 30 min to eliminate ethanol.
cDNA labeling with a reaction fluorescence dye Prepare the amine-modified cDNA. Thoroughly dissolve the amine-modified DNA in 5 μl of nuclease-free water, warm in a 42°C water bath for 5 min. Prepare labeling buffer: 2.5 g sodium bicarbonate in 100 ml water. Add 3 μl labeling buffer to the amine-labeled cDNA. Dissolve one vial of dye in 2 μl of high quality DMSO, vortex for 10 s. Add the 8 ul amine-modified cDNA into the dissolved dye. Incubate the reaction in dark for 1 h.
Purification of dye-labeled cDNA using QIAquick PCR Purification column Add 8 μl of nuclease-free water and 2 μl 3 M sodium acetate pH 5.5 to the reaction mixture. Add 100 μl buffer PB to the mixture. Apply the sample to the QIAquick column and centrifuge for 60 s. Discard flow-through and place the QIAquick column back into the same tube. To wash, add 0.75 ml buffer PE to the column and centrifuge for 60 s. Centrifuge in a 2 ml collection tube for 1 min. Place QIAquick column in a clean 1.5 ml tube. Add 50 μl buffer EB and centrifuge for 1 min.
Hybridization protocol
Prepare hybridization samples 1. Add 20 μl water to dissolve the dried labeled-cDNA (600ng). 2. Add 5 μl 10 × Blocking Agent 3. Add 25 μl 2 × GE × Hybridization Buffer H1-RPM. Mix well by pipetting to reduce bubble formation. Spin for 1 min and place on ice.
Prepare the hybridization assembly 1. Load a clean gasket slide into the Agilent Suretlyb chamber base with the label facing up. 2. Slowly dispense 40 μl hybridization samples onto the gasket well. To minimize bubble formation. 3. Slowly place an array “active side (Agilent facing down)” onto the gasket slide. 4. Place the SureHyb chamber clamp cover on the sandwiched slides and slide the clamp assembly onto both pieces. 5. Hand-tighten the clamp on to the chamber. Rotate the gasket slide when tightening. 6. Vertically rotate the assembled chamber to wet the gasket and assess the mobility of the bubbles. 7. Place assembled slide chamber in a hybridization oven set to 65°C, 15 rpm. 8. Hybridize at 65°C for 17 h.
Washing This step is performed under dark conditions. 1. Add triton into the X-102 to Gene Expression washing buffer 1 and 2. 2. Pre-warm gene expression wash buffer 2 in a 37°C water bath O/N 3. Dessemble the hybridization chamber, put gaskets and slide into Washing Buffer 1. Use a tweezer to separate the slide from the cover slide. 4. Put the slide into the washing container and array side facing out. Wash for 1 min 5. Transfer the slide into another container containing Washing Buffet 2 and wash for another min. 6. Remove the slide slowly to minimize the attachment of water. Put the slide into a 50 ml tubes in dark places.
Scan protocol
Slides were scanned using an Agilent Scanner (Agilent technologies, Santa Clara, CA, USA) at 5-µm resolution. All slides were scanned using 100% laser power; PMT voltages were automatically adjusted using the Genepix Pro 6.0 software acquisition system to obtain maximal signal intensities with <0.02% probe saturation. The resulting 16 bit images were processed using the GenePix Pro 6.0 image analysis software (v6.0.1.26).
Data processing
Microarray data normalization Microarray normalization is carried out using maanova function in R program version 2.2.1. Eight arrays in one slide were globally normalized using glowess method. 1. library(maanova) go to the directory: setwd("C:\\Documents and Settings\\Wang\\Desktop\\Erwinia in vivo\\rcsC MBMA") 2. Read data: Data = read.madata("NormInput.txt", designfile="Designfile1.txt", header=TRUE, spotflag=TRUE, metarow=1, metacol=2, row=3, col=4, geneID=5, pmt=6) 3. Log transformation: LogData = createData(Data, n.rep=1, log.trans=TRUE) 4. Normalization by Glowess method: NormalData=transform.madata(LogData, method=c("glowess")) 5. Save the plot files postscript(file="PlotRawData.ps") riplot(LogData, onScreen=FALSE) postscript(file="PlotGLOWESS.ps") riplot(NormalData, onScreen=FALSE) 6. close the graph: graphics.off() norm.temp<-cbind(NormalData$metarow, NormalData$metacol,NormalData$row, NormalData$col, NormalData$geneID, NormalData$data) 7. output: write.table(norm.temp, file="Normal_Data.csv", sep=",", row.names=FALSE, col.names=TRUE, quote=FALSE)
Data selection Statistical comparisons were performed using multiple testing procedures to evaluate statistical significance for differentially expressed genes. A modified t-test (p-value) was computed to measure the significance associated with each differential expression value. A gene expression value was decided to be significantly different in the mutant and over-expression strains when the p-value was less than 0.05 (except otherwise mentioned) and the expression ratio was ≥ 2.0or ≤ 0.5. Gene functions were assigned using data from EcoCyc (http://ecocyc.org/).
