Slides were spun dry and scanned using a microarray dual channel laser scanner (ScanExpress Lite da PerkinElmer®, USA) at 10 μm resolution, 100% laser power and PMT levels which were adjusted in order to obtain similar distributions of red and green signal intensities.
For each time point, gene expression analysis was done based on information obtained from four slides, one dye-swap pair for each biological replicate. ScanArray Express (PerkinElmer®) software was used to generate the slide images and raw intensity data which were then analyzed using specific packages from the R statistical language. Spots of good quality (positive flag value) were considered to be analyzed. Data were inspected for spatial biases on both red and green channels (background and signal), for print-tip bias, dye bias, and bias dependent of intensity using the LIMMA and marray packages. Background correction was done with normexp method. Robust spline and aquantile methods were used for normalization within and between arrays, respectively. A linear model that incorporates biological and technical replication was used for statistical analysis. A list of differentially expressed genes was generated by applying adjusted p-values for multiple tests using ‘BH’ method, which works with the expected proportion of false-positives (FDR- False Discovery Rate) among the rejected hypothesis. Graphics were drawn using GraphPad version 5.03. Heatmap and cluster graphics were created using gplots package and personal scripts (R statistical language).
VALUE = normalized log2 Fold-change (irradiated samples/control)
Cultures with 5×108 parasites in 20 mL of LIT medium (2x107 cells/mL) were exposed to a dose of 500 Gy (1578 Gy/h per 20 minutes) in a cobalt (60Co) irradiator located at Centro de Desenvolvimento da Tecnologia Nuclear (CDTN), Belo Horizonte, Brazil
Epimastigote cells were subjected to RNA extraction using Trizol reagent (Invitrogen Life Technologies, USA) and RNA samples were purified using RNeasy® MiniEluteTM Cleanup Kit (Qiagen, German) according to the manufacturers’ instructions. Total RNA was quantified using a Nanodrop ND-100 UV/Vis spectrophotometer (NanoDrop Technologies, USA) and the overall RNA quality was assessed by denaturing gel electrophoresis . Total RNA (2 μg) was amplified using the Amino Allyl MessageAmp II kit (Ambion, USA), according to the manufacturer’s specifications.
Epimastigote cells were cultivated at 28 ◦C in LIT medium (Liver Infusion Tryptone - liver digest neutralized) supplemented with complement-inactivated 10% fetal bovine serum, streptomycin sulfate (0.2 g/L), and penicillin (200,000 units/L).
Slides were pre-hybridized by placing them in coupling jars containing pre-hybridization solution (5× SSC, 0.1% SDS, 1%BSA) at 42 °C for one hour. Slides were washed twice by immersing 10 times in a beaker containing MilliQ water followed by dipping three times in isoamyl alcohol, and were subsequently spun dry. The slides containing 30 µL of samples in hybridization buffer were hybridized for 14 hours in a water bath at 42° C in the dark under cover slips inside Corning® hybridization chambers (Corning, USA). Slides were then washed two times for five minutes each in a low stringency wash solution (2× SSC, 0.1% N-lauroysarcosine) at 42 °C (first wash) and RT (second wash), followed by two washes of five min in medium stringency wash (0.1× SSC, 0.1% N-lauroysarcosine) at RT and two washes for five minutes each in high stringency wash solution (0.1× SSC) at RT.
Aminoallyl amplified RNA was labeled with Cy3 and Cy5 according to a modified version of the AminoAllyl MessageAmp II Kit (Ambion, USA) and TIGR's standard operational procedure – SOP #M008 (ftp://ftp.jcvi.org/pub/data/PFGRC/MAIN/pdf_files/protocols/M008.pdf). Briefly, we followed the manufacturer’s instructions for the labeling step, but the initial amount of amplified RNA was changed to 8 μg. The Cy3 and Cy5 labeled samples were then combined. Labeled RNA was purified away from unincorporated dyes using YM-30 Microcon columns following manufacturer's specifications (Millipore®, USA). The final sample was dried again and resuspended in 30 µL of hybridization buffer (50% formamide, 5× SSC, 0.1% SDS, 0.1M DDT, and 6% salmon sperm as blocking agent) according to TIGR's SOP #M008. The solution was heated to 95 oC during 3 minutes and placed in ice for 30 seconds. After a brief centrifugation, the solution was dispensed onto the slide surface and covered with a coverslip.