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Materials and Methods, Half Life Measurements and Preparation of RNA Samples Half lives. were determined as described by Seeley et al 5 with the. following modifications Arabidopsis thaliana ecotype Columbia. were grown on plates containing 1 Murashige and Skoog salts. 1 Gamborg s vitamins and 1 sucrose for 2 weeks at 22 C and. 16 h 8 h light dark cycles The plants were then transferred to. a flask with incubation buffer 5 After a 30 min incubation. 3 deoxyadenosine cordycepin was added to a final concen. tration of 0 6 mM time 0 Tissue samples were harvested at. regular intervals thereafter and quickly frozen in liquid nitrogen. Total RNA was isolated and analyzed by Northern blot using. standard techniques Cordycepin was used to inhibit transcrip. tion in these studies because its use is routine in plants 5 25 in. contrast to other inhibitors such as alpha amanitin and is more. effective in leaf tissue than Actinomycin D 25 presumably. because of poor penetration, Hybridization of cDNA Microarrays The 11 521 element cDNA. microarray print name MSU 2 03 00 prepared by the Arabi. dopsis Functional Genomics Consortium was used in all exper. iments 26 One hundred micrograms of total RNA correspond. ing to time 0 and 120 min after cordycepin treatment was labeled. during first strand cDNA synthesis with Cy3 and Cy5 labeled. dUTP respectively as described 26 Three independent. cordycepin treatments biological replicas were performed and. Fig 1 Strategy for monitoring mRNA stability using cDNA microarrays a. RNA samples were isolated Each pair of samples from the 0 and RNA samples corresponding to 0 and 120 min after the addition of the. 120 time points was used in two microarray hybridizations the transcriptional inhibitor cordycepin were labeled with Cy3 and Cy5 respec. second with reverse labeling relative to the first technical tively and used to hybridize 11K microarray slides Each pair of RNA samples. replicas Measurement of the fluorescence corresponding to was reverse labeled for a separate microarray hybridization These hybridiza. hybridization intensities was performed with the ScanArray 4000 tions were performed with samples from three independent cordycepin treat. Microarray Acquisition System Packard BioChip Technologies ments for a final data set of six slides Half life values were then estimated. Billerica MA We used the SCANALYZE V2 44 software from the normalized ratios b Nonreproducible spots decrease as a function. of the number of slides nearly leveling out when the data from four slides is. http rana lbl gov EisenSoftware htm to extract the infor. combined The quality of the slides best to worst or worst to best based on the. mation of the images generated The raw data for these exper extent of visible gradients see Materials and Methods does not significantly. iments is available from the Stanford Microarray Database affect the reproducibility of the data when two or more slides are considered. SMD http genome www stanford edu microarray ref although the curves are slightly steeper with better slides. 27 ExptID 11374 11333 11339 11323 11375 and 11342, Microarray Data Analysis Stringent quality control measures were EST identification the BLASTN program was used to search the. applied to define the working data set Spots with abnormal completed Arabidopsis genome sequence downloaded from The. shapes or high local background were discarded manually Spots Institute for Genomic Research TIGR Functional categories. with channel intensity values smaller than the mean plus two were obtained from the Munich Information Center for Protein. standard deviations of each slide background or with GTB2 Sequences For analysis of gene expression data across multiple. values smaller than 0 65 in more than two channels were experiments the CLUSTER and TREEVIEW software were used. discarded because of low signal The quality of the hybridization ref 28 http rana lbl gov EisenSoftware htm Visual im. was also evaluated by visual inspection of the gradients using the ages were generated with the TREEVIEW software by using the. most sensitive setting of the Array Color Plot tool imple output generated by the hierarchical clustering program of the. mented in SMD Slides that showed gradients in more than 25 CLUSTER software The uncentered correlation similarity metric. of the array surface and or that had R2 values 0 15 indicating was used to perform average linkage clustering. a strong dependence on spatial location were not used for data Sequences of AtGUTs 5 UTR coding sequence 3 UTR. analysis The percent of the array surface that exhibited gradi were obtained from the TIGR Arabidopsis genome as described. ents was also used to order the slides from worst to best or best in the supporting information which is published on the PNAS. to worst in Fig 1 web site www pnas org Frequencies of overlapping 1 bp win. The Z score method in log space with a 90 trimmed data set dow oligonucleotides up to 6 nt in length were determined in. was used for global normalization of the data 26 The differ the 3 UTR sequences of AtGUTs and also in the 3 UTR. ence in mRNA levels between the time points considered 0 and sequences of a control set of genes as described by van Helden. 120 min can be used to estimate the rates of decay by using the et al 29 The control set was derived from 4 064 clones. equation ln Normalized Ratio kdecayt with the half life corresponding to stable transcripts on the array To assess. being t1 2 0 693 kdecay because mRNA degradation generally significance 1 000 random samples of the same size as the test. obeys first order kinetics 24 Statistical analysis of the ratios set were taken from the control sequences and oligonucleotide. was performed using the t test as described in the text frequencies were determined in these samples The criteria for. significance were as follow i oligonucleotide was at least 2 fold. Sequence and Gene Expression Analysis Sequences of 59 clones more abundant in unstable than in whole control set ii. representing Arabidopsis thaliana genes for unstable transcripts oligonucleotide had frequencies 2 SD above mean frequency. Downloaded by guest on September 30 2020, AtGUTs were determined and found to be consistent with in the 1 000 random samples from control set iii oligonucle. sequences deposited in GenBank For expressed sequence tag otide was present in 10 of test sequences Additional MEME. 11514 www pnas org cgi doi 10 1073 pnas 152204099 Gutie rrez et al. Fig 2 Confirmation of the instability of transcripts identified by microarray analysis a Half life values determined by Northern blot are comparable to. estimates from microarray analysis for four randomly selected unstable transcripts Northern blot values are representative of at least three independent. cordycepin time courses b Representative Northern blot analysis of cordycepin time courses for two randomly selected unstable and two stable transcripts. Samples consisted of 10 g of total RNA isolated from the indicated time points c Quantitation of the decrease in mRNA abundance and half life estimation. The signal for eIF 4A does not change significantly during the time courses and was used as a reference for equal loading. searches 30 were carried out as described at http meme correlated across multiple experiments data not shown Based. sdsc edu Various combinations of the MEME parameters were on this criterion the selected clones corresponded to 100 genes. tested motif distribution 1 3 number of motifs 3 5 motif that were termed Arabidopsis thaliana genes with unstable. width 5 50 Programs written in the Practical Extraction and transcripts AtGUTs because the calculated half lives of the. Report Language Perl were used for sequence extraction and encoded transcripts were 60 min or less see Table 2 which is. manipulation published as supporting information on the PNAS web site. Similarly we identified 225 genes with moderately unstable. Results and Analysis mRNAs whose estimated half lives ranged from 60 to 120 min. Monitoring mRNA Stability by Using cDNA Microarrays mRNA decay The great majority of the transcripts in Arabidopsis appeared to. rates expressed as half life values are typically measured by decay with rates greater than 2 h consistent with the idea that. monitoring the disappearance of a transcript by Northern blot most messages in plants are relatively stable 1. after transcription of the corresponding gene has been halted The 11K microarray used in these studies represents an. We combined this simple experimental strategy with the highly estimated 7 800 unique genes 26 so the 100 AtGUTs we. parallel power of DNA microarray analysis 31 as outlined in identified correspond to about 1 This number likely repre. Fig 1a Total RNA samples corresponding to 0 and 120 min sents an underestimate of unstable Arabidopsis transcripts es. time points after transcriptional inhibition with cordycepin were pecially when extrapolated to the whole genome for several. isolated One hundred micrograms of total RNA from each of reasons First unstable mRNAs are often associated with low. these samples was used to synthesize cDNA probes by incorpo steady state levels which may be underrepresented in the EST. rating Cy3 or Cy5 labeled dUTP during oligo dT primed re collections used for the 11K microarray Second some unstable. verse transcription The probes were combined and used for transcripts likely fall below the detection limits of the microarray. hybridization of the 11 521 elements cDNA microarray 11K technique or might not meet our stringent reproducibility re. microarray prepared on glass slides by the Arabidopsis Func quirements However the channel intensity distribution for. tional Genomics Consortium We performed three biological AtGUTs resembled that of the whole array suggesting the. replica experiments each with a reverse labeling technical rep AtGUTs identified were not strongly biased to either high or low. licate The purpose of these repetitions was to increase the expression levels on the 11K microarray The identification of. likelihood of detecting significant differences in mRNA levels highly expressed AtGUTs was an added bonus from this analysis. while decreasing the likelihood of false positives which might be because these transcripts should greatly facilitate future studies. common on microarray studies with one or two slides 32 Quite of steps in their degradation Finally multiple members of. reasonably the number of nonreproducible normalized intensity closely related gene families may be missed because the 11K. ratios 2 decreased as a function of the number of slides nearly microarray was not designed to resolve gene family members. leveling out below 5 when four slides were considered Fig Thus on the basis of our work it seems valid to estimate that at. 1b Based on this data and to be rigorous we defined our least 1 of the genes of Arabidopsis correspond to unstable. working data set as all those clones with reproducible normalized transcripts. intensity ratios 2 in five of six slides We used conventional Northern blot analysis of cordycepin. time courses with several time points to confirm the 11K. At Least 1 of Clones on the 11K Arabidopsis Microarrays Correspond microarray data Four randomly selected transcripts with half. to Unstable Messages To identify and characterize the most lives of less than 60 min showed comparable turnover rates in full. unstable transcripts from our working data set we focused our cordycepin time courses Fig 2a Two representative examples. attention on the transcripts that were most diminished after are shown in Fig 2 b and c In addition we assessed the statistical. treatment with cordycepin for 120 min Clones whose median significance of the ratio values for the genes of interest When. normalized intensity ratios were 4 0 vs 120 min and that met using the t test and the conservative Bonferroni method to adjust. PLANT BIOLOGY, several quality control criteria see Materials and Methods were P values 33 all selected AtGUTs showed significantly different.
used for further analysis In this study ESTs that overlap with the ratios from the mean of the population at 0 0001 see Table. same annotated ORF were considered as representing the same 2 Several genes with moderately unstable messages according. Downloaded by guest on September 30 2020, gene This is a reasonable assumption because the expression to the microarray studies were at least moderately unstable in. patterns of groups of ESTs that match the same ORF were well Northern blots data not shown Four stable transcripts accord. Gutie rrez et al PNAS August 20 2002 vol 99 no 17 11515. Fig 3 Instability is associated with a broad range of plant processes Genes with unstable transcripts were classified according to the scheme of Munich. Information Center for Protein Sequences AtGUTs are predicted to participate in a broad range of cellular processes with transcriptional functions over. represented compared with what is expected based on the whole genome annotation To allow comparison AtGUTs were classified based on the information. released for the annotation of the whole A thaliana genome sequence in December 2000 The most updated annotation for the AtGUTs is also included May. 2002 although a whole genome annotation based on this updated information is not yet available. ing to microarray data were also found stable by Northern blot More than half of the AtGUTs could be assigned to a Munich. analysis further validating our results Two representative ex Information Center for Protein Sequences category see Table. Department of Energy Plant Research Laboratory and Delaware Biotechnology Institute Department of Plant and Soil Sciences and College of Marine Studies University of Delaware 15 Innovation Way Newark DE 19711 E mail green dbi udel edu www pnas orgcgidoi10 1073pnas 152204099 PNAS August 20 2002 vol 99 no 17 11513 11518 Downloaded at Microsoft Corporation on November 17 2019

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