4.4 Fase III: Gestión de emergencias 4.4.5 Diseño y ejecución de simulacro interno Bowtie aligned sequences from Zhang et al. (2012) were extracted from GEO reposi- tory (Edgar et al., 2002) (ascension numbers: GSM847326, GSM847327, GSM847329, GSM847329, GSM847330) and converted into BAM format using SAMTools v0.1.18- dev (Li et al., 2009). HOMER v4.1 (Heinz et al., 2010) was used to extract hypersen- sitive sites in BED format individually for each of the 5 samples using Arabidopsis v1.3 (TAIR10) reference provided by HOMER. A custom parallel application was written in C to scan for presence or absence of a motif in promoters of Arabidopsis genome (TAIR10). A motif was deemed to be present if it matched exactly one of the sequences representing the motif, which is also included as part of the motif output from MeMe. The locations of sites matching MeMe motifs were checked for overlap with hypersensitive sites for each sample using a custom Perl script. Wellington, a novel tool for analysing DNase data (Jason Piper, unpublished) was used to extract information about the starts or end of the sequencing read, where DNase has broken the double stranded DNA, and plot them using “matrix2png” package for Python, as well as plot average cut profile using “averageProfilePlotter” Python script (courtesy of Jason Piper). The colour of the spots in DNase figure represents the strand the cut is located: red - positive strand, green - negative. The brightness of the spot on a DNase image represents mumber of cuts, reads starting or ending at that position, brighter the spot, larger number of reads it represents. 4.2.3 Plant growth Arabidopsis seeds were stratified in 0.1% w/v agarose at 4◦C for 72 h in complete darkness. Stratified seeds were sown in pre-watered Arabidopsts soil mix (6:1:1 ratio of Levington F2 compost:sand:vermiculite) containing Intercept (Everris, Ipswich) in 4-cm pots (P24, Plant-pak). Pots were converted with cling film and placed in a growth chamber to germinate. The covering was removed 7 days post sowing and seedlings thinned out to give one plant per pot. Plants were grown in standardised conditions under 16 h light 18 h dark days at 20◦C, 350 ppm CO2 concentration and 120µmol m2s−1 light. 4.2.4 Fungal growth Botrytis cinerea(Botrytis) strain pepper spores (Denby et al., 2004) were germinated and cultured on sterile tinned apricot halves (Tesco, UK) in petri dishes 4 weeks prior to use. Two weeks prior to use, Botrytis was sub-cultured using the same procedure. Sub-cultures were incubated at 25◦C in complete darkness. Spores were harvested in sterile water and filtered through glass wool to remove hyphae. Inoculums were prepared by suspending spores in half strength sterile grape juice (Tesco, UK) at a concentration adjusted to 1×105 spores/ml. Spores concentration was measured with a hemocytometer. 4.2.5 Phenotype Analysis Plant leaves inoculated with Botrytis were grown in 3 trays (10-15 leaves per line in each tray) at 90% humidity level in sealed trays in 12 h day/night cycles. Images were taken of the trays with the leaves for phenotypic analysis at 48 h, 57.5 h and 72 h post infection. Area around each fungus in every leaf was manually draw and calculated using ImageJ (Schneider et al., 2012) software. The calculated areas were analysed using one-sample Kolmogorov-Smirnov test for normality, Table C.1. The areas for each samples were normalised using mean and standard deviation within the sample. All samples were approximately derived from a normal distribution and therefore, t-test was used to determine whether there was statistical difference between each sample and control (Col0). 4.2.6 Microarray analysis RNA extraction Snap frozen Arabidopsis leaves were ground in 1 ml Trizol reagent (Invitrogen, Pais- ley) using a Dremel drill for 1 min until the sample was completely homogenised. The drill-bit was frozen in liquid nitrogen prior to use to prevent thawing of leaf tis- sue. Samples were incubated at room temperature for 5 min to allow for dissociation of nuclear protein complex before adding 200µl chloroform. Reaction was shaken vigorously by hand for 15 s and incubated for a further 3 min at room tempera- ture. Samples were centrifuged at 8000 ×g for 15 min at 4◦C. The upper aqueous phase (60% of the volume) was transferred to a fresh 1.5 ml Eppendorf tube fol- lowed by addition of 0.5 ml of isopropanol to precipitate the RNA. Samples were mixed by inverting tubes several times and incubated at -20◦C for 2 h. Samples were centrifuged at 8,000 ×g for 20 min at 4◦C. RNA pellets were washed with 1 ml of 75% EtIH followed by centrifugation at 8,000 ×g for 10 min at 4◦C. The supernatant was completely removed and pellet allowed to air-dry for 5 min before re-suspension in 100µl RNase free water. Total RNA was purified using Qiagen RNeasy purification kit (Qiagen, Manchester) according to the manufacturer’s in- structions, except for the final step where purified RNA was eluted from the column with 2 x 40µl RNase free water. Total RNA concentration was measured using a Nanodrop ND-1000 spectophotometer (Thermo-Scientific, Nortumberland) using 1µl sample. Total RNA quality was determined using a 2100 Bioanalyser with the RNA 6000 Nano LabChip kit according to the manufacturer’s instructions (Agilent). The Bioanalyser assesses total RNA integrity by measuring the 18S and 28S rRNA peaks using high-resolution electrophoresis system. Where total RNA samples dis- played no rRNA peaks or a poor 18S/28S ratio (<1), total RNA was isolated from alternative leaf samples. RNA amplification Total RNA was amplified using MessageAmp-II aRNA Amplification Kit (Invit- rogen, Paisley) according to the manufacturer’s instructions, using a single round of amplification and anin vitro transcription step and an incubation time of 14 h. The quality of amplified RNA was determined using a 2100 Bioanalyser with the RNA 6000 Nano LabChip kit according to the manufacturer’s instructions (Agilent, Wokingham) and concentration of the purified sample was measured using a Nan- odrop ND-1000 spectrophotometer. Good quality amplified RNA should display a size distribution that is approximately a normal distribution (bell shaped) Where the size distribution was clearly abnormal or the amplified RNA concentration was <300 ngµl−1, total RNA for that sample was re-amplified. Microarray experimental design Erf plants infected with Botrytis were compared to Col-0 plants also infected with Botrytis 24 h post infection. RNA from 4 biological replicates were pooled after amplification for mutant and wild type leaves. Comparisons were made using a total of 4 technical replicates: 2 replicates for one set of dyes, then 2 replicates after dyes were swapped. In the protoplast experiment overexpression vectors forERF14 and ESE1 were compared to protoplasts where no vector was added, but otherwise went through the same process. For each sample, RNA from 4 technical replicates were pooled after amplification. Comparisons were made using a total of 4 technical replicates after pooling: 2 replicates for one set of dyes, then 2 replicates after dyes were swapped. Direct labelling of amplified RNA Approximately 5µg of pooled amplified RNA, generated by combining equal amounts of amplified RNA from each of the appropriate biological, or technical replicates in case of protoplasts, was combined with 0.5µl of random nanomer (3µg ml−1) (In- vitrogen, Paisley) and 0.5µl of RNase inhibitor (RNase OUT; Invitrogen)for total volume of 10.5µl. Samples were incubated at 70◦C in a thermocycler for 10 min. Superscript mastermix was created by combining the following reagents per reac- tion: 4µl 5×Superscript II First Strand Buffer (Invitrogen, Paisley), 2µl 0.1 mol DTT (invitrogen), 1µl dNTP mix (10 mmol dATP, 10 mmol dCTP, 10 mmol dGTP, 10 mmol dTTP) and 1µl Superscript II reverse transcriptase (Invitrogen, Paisley). Samples were labelled by adding 8µl of superscript mastermix with 1.5µl of either Cy3- or Cy5-dCTP (GE Healthcare, Chalfont St Giles) followed by incubation in the dark at 42◦C for 2.5 h. 2µl of 2.5 mol NaOH was added to each of the labelled cDNA samples followed by incubation at 37◦C for 15 min. Samples were combined with 10µl of 2 mol MOPS buffer and purified using QiaQuick PCR purification kit (Qiagen, Manchester) according to the manufacturer’s instructions. At the end of the procedure, the purified cDNA was eluted with 2×30 µl of Buffer EB (Qiagen, Manchester). The concentration of purified sample was measured at 532 nm (Cy3) or 635 nm (Cy5) wavelength using nanodrop ND-1000 spectrophotometer. Samples were kept in the dark throughout labelling steps to minimise light degradation. CATMA array hybridisation CATMAv4 (Hilson et al., 2004) array slides were prepared for hybridisation by incubating them in Coplin jars with Pre-Hybridisation buffer (1.2g Bovine Serum Albumin (Sigma-Aldrich, A9418), 5×SSC, 0.1% SDS in 120 ml sterile water) (pre- warmed to 42◦C in an air incubator) for 1 h. CATMA array slides were washed by submerging in sterile water for a total of 5 washes and a final wash with isopropanol. Slides were dried by centrifugation for 1 min at 2000×g. Combinations of up to 40 pmol of the appropriate Cy3- or Cy5-labelled samples were freeze dried until nearly dry and resuspended in 50µl of hybridisation buffer (12.5µl Formidem, 12.5µl 20×SSC, 0.35µl of 14% SDS, 6.25µl of 4µg ml−1 Yeast tRNA (Invitrogen, Paisley) and 18.4µl sterile water). Resuspended samples were incubated at 95◦C for 5 min in a thermocycler followed by centrifugation at 10000×gfor 1 min. The hybridisation mix was applied to an array slide located within hybridisation chamber (Corning, Corning) followed by the application of a coverslip (Sigma Aldrich, Gillingham) and chamber cover. Hybridisation chambers were placed in a humid environment at 42◦C for 16 h. Coverslips were removed by submerging array slides in 250 ml of Wash Solution 1 (2×SSC, 0.07% SDS and 250 ml sterile water) (preheated to 42◦C in an air incubator) until free. Hybridised slides were then incubated in Wash Solution 1 in a hybridisation rack for 5 min with gentle shaking. Slides were then incubated in 250 ml of Wash Solution 2 (0.1×SSC, 0.07% SDS, 250 ml sterile water) for 10 min with gentle shaking. Then slides were incubated in Wash Solution 3 (0.1×SSC and 995 ml of sterile water) for 1 min with gentle shaking for a total of 4 washes. Finally, slides were briefly immersed in isopropanol and dried by centrifugation at 1000×g for 1 min. 4.2.7 Microarray scanning Array slides were scanned on Affymetrix slide scanner with default settings. The data extracted from scanned images was quantified using Imagene 7.5 software (BioDiscovery, Inc.) (M´edigue et al., 1999). 4.2.8 Expression Analysis Comparisons between mutant and wild-type samples were analysed using R (Bio- conductor) (Gentleman et al., 2004) package limmaGUI (Wettenhall and Smyth, 2004). Raw data was normalised within arrays using PrintTip lowess transforma- tion and then normalised between arrays using aquantile-normalisation. The data was fitted with a linear model using the least squares method. P-values were ad- justed for multiple testing using Benjamini and Hochberg method with threshold of 0.05 to control for false discovery rate. In document Elaboración de un plan integral de gestión de riesgos laborales en la planta procesadora de quinua Maquita ubicada en Calpi, provincia de Chimborazo (página 122-128)
