The balance of reproducibility, sensitivity, and specificity of lists of differentially expressed genes in microarray studies

Shi, Leming; Jones, Wendell; Jensen, Roderick V.; Harris, Stephen; Perkins, Roger; Goodsaid, Federico; Guo, Lei; Croner, Lisa J.; Boysen, Cecilie; Fang, Hong; Qian, Feng; Amur, Shashi; Bao, Wenjun; Bárbácioru, Cátálin; Bertholet, Vincent; Cao, Xiaoxi; Chu, Tzu Ming; Collins, Patrick J.; Fan, Xiao Hui; Frueh, Felix W.; Fuscoe, James C.; Guo, Xu; Han, Jing; Herman, Damir; Hong, Huixiao; Kawasaki, Ernest S.; Li, Quan Zhen; Luo, Yuling; Ma, Yunqing; Mei, Nan; Peterson, Ron L.; Puri, Raj K.; Shippy, Richard; Su, Zhenqiang; Sun, Yongming; Sun, Hongmei; Thorn, Brett T.; Turpaz, Yaron; Wang, Charles; Wang, Sue Jane; Warrington, Janet A.; Willey, James C.; Wu, Jie; Xie, Qian; Zhang, Liang; Zhang, Lu; Zhong, Sheng; Wolfinger, Russell D.; Tong, Weida

doi:10.1186/1471-2105-9-s9-s10

Cited by 212 publications

(169 citation statements)

References 54 publications

(59 reference statements)

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“…Genomics approaches continue to be criticized because of inconsistent results (Miklos and Maleszka 2004;Draghici et al 2006), and while some of this inconsistency arises from data processing (Shi et al 2008), differences in sample RNA integrity markedly affect transcript quantification and likely are the cause of much of the inconsistency (Auer et al 2003;Lipska et al 2006;Atz et al 2007;Thompson et al 2007;Popova et al 2008). Sample RNA of the highest quality is therefore imperative.…”

Section: Discussionmentioning

confidence: 99%

Improved RNA preservation for immunolabeling and laser microdissection

Brown

Dw²

2009

RNA

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Microdissection techniques have the potential to allow for transcriptome analyses in specific populations of cells that are isolated from heterogeneous tissues such as the nervous system and certain cancers. Problematically, RNA is not stable under the labeling conditions usually needed to identify the cells of interest for microdissection. We have developed an immunolabeling method that utilizes a high salt buffer to stabilize RNA during prolonged antibody incubations. We first assessed RNA integrity by three methods and found that tissue incubated in high salt buffer for at least 20 h yielded RNA of similar quality to that for RNA extracted from fresh-frozen tissue, which is considered highest quality. Notably, the integrity was superior to that for RNA extracted from tissue processed using rapid immunolabeling procedures (5 min total duration). We next established that high salt buffer was compatible with immunolabeling, as demonstrated by immunofluorescent detection of dopamine neurons in the brain. Finally, we laser microdissected dopamine neurons that were immunolabeled using high salt buffer and demonstrated that RNA integrity was preserved. Our described method yields high quality RNA from immunolabeled microdissected cells, an essential requirement for meaningful genomics investigations of normal and pathological cells isolated from complex tissues.

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Section: Discussionmentioning

confidence: 99%

Improved RNA preservation for immunolabeling and laser microdissection

Brown

Dw²

2009

RNA

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“…Student's t test was performed in group-wise comparison of gene expression. A P value < 0.05 combined with a greater than twofold change were considered significant in accordance with the recommendations of the MicroArray Quality Control project group, which were shown to achieve a balance of sensitivity, specificity, and reproducibility (38,39).…”

Section: Methodsmentioning

confidence: 99%

Paneth cell marker expression in intestinal villi and colon crypts characterizes dietary induced risk for mouse sporadic intestinal cancer

Wang

Peregrina

Dhima

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Nutritional and genetic risk factors for intestinal tumors are additive on mouse tumor phenotype, establishing that diet and genetic factors impact risk by distinct combinatorial mechanisms. In a mouse model of dietary-induced sporadic small and large intestinal cancer in WT mice in which tumor etiology, lag, incidence, and frequency reflect >90% of intestinal cancer in Western societies, dietary-induced risk altered gene expression profiles predominantly in villus cells of the histologically normal mucosa, in contrast to targeting of crypt cells by inheritance of an Apc 1638N allele or homozygous inactivation of p21 Waf1/cip1 , and profiles induced by each risk factor were distinct at the gene or functional group level. The dietary-induced changes in villus cells encompassed ectopic expression of Paneth cell markers (a lineage normally confined to the bottom of small intestinal crypts), elevated expression of the Wnt receptor Fzd5 and of EphB2 (genes necessary for Paneth cell differentiation and localization to the crypt bottom), and increased Wnt signaling in villus cells. Ectopic elevation of these markers was also present in the colon crypts, which are also sites of sporadic tumors in the nutritional model. Elevating dietary vitamin D 3 and calcium, which prevents tumor development, abrogated these changes in the villus and colon cells. Thus, common intestinal cancer driven by diet involves mechanisms of tumor development distinct from those mechanisms that cause tumors induced by the rare inheritance of a mutant adenomatous polyposis coli (Apc) allele. This is fundamental for understanding how common sporadic tumors arise and in evaluating relative risk in the population.sporadic | colon cancer | Western diet | microarray

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“…Because microarrays can be very reliable for identifying differentially expressed genes when fold changes are generally large (Shi et al, 2008), as in this study, we did not consider it essential to evaluate changes in gene expression by quantitative real-time PCR using the same samples. In future studies, it will certainly be of interest to examine the expression of key genes identified in this study under a variety of different conditions.…”

Section: Many Genes Are Differentially Expressed In Y831f Plantsmentioning

confidence: 99%

“…Thus, all three of these critical growth-associated genes were altered in expression in a manner that would predict increased growth of the Y831F mutant plants. Only IPS1 is regulated by BR signaling in wild-type plants (Table III), but even with this gene the expression is modulated to a greater extent in the Y831F plants.Because microarrays can be very reliable for identifying differentially expressed genes when fold changes are generally large (Shi et al, 2008), as in this study, we did not consider it essential to evaluate changes in gene expression by quantitative real-time PCR using the same samples. In future studies, it will certainly be of interest to examine the expression of key genes identified in this study under a variety of different conditions.…”

mentioning

confidence: 99%

Enhancing Arabidopsis Leaf Growth by Engineering the BRASSINOSTEROID INSENSITIVE1 Receptor Kinase

Sun

et al. 2011

Plant Physiology

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The BRASSINOSTEROID INSENSITIVE1 (BRI1) receptor kinase has recently been shown to possess tyrosine kinase activity, and preventing autophosphorylation of the tyrosine-831 regulatory site by site-directed mutagenesis enhances shoot growth. In this study, we characterized the increased leaf growth of Arabidopsis (Arabidopsis thaliana) plants expressing BRI1(Y831F)-Flag compared with BRI1-Flag (both driven by the native promoter and expressed in the bri1-5 weak allele background) and provide insights into the possible mechanisms involved. On average, relative leaf growth rate was increased 16% in the Y831F plants (in the bri1-5 background), and the gain of function of the Y831F-directed mutant was dominant in the wild-type background. Leaves were larger as a result of increased cell numbers and had substantially increased vascularization. Transcriptome analysis indicated that genes associated with brassinolide biosynthesis, secondary cell wall biosynthesis and vascular development, and regulation of growth were altered in expression and may contribute to the observed changes in leaf architecture and whole plant growth. Analysis of gas exchange and chlorophyll fluorescence indicated that Y831F mutant plants had higher rates of photosynthesis, and metabolite analysis documented enhanced accumulation of starch, sucrose, and several amino acids, most prominently glycine and proline. These results demonstrate that mutation of BRI1 can enhance photosynthesis and leaf growth/vascularization and may suggest new approaches to increase whole plant carbon assimilation and growth.Brassinosteroids (BRs) are essential plant steroid hormones that regulate multiple aspects of growth and development, including cell elongation, cell division, vascular differentiation, seed germination, timing of senescence, male fertility, and organ formation (Clouse and Sasse, 1998; Altmann, 1999;Nakaya et al., 2002;Gonzalez et al., 2010). It is known that BRs bind to the BRASSINOSTEROID-INSENSITIVE1 (BRI1) receptor kinase, which functions in conjunction with the coreceptor BRASSINOSTEROID-ASSOCIATED KINASE1 (BAK1) in hormone perception and signal transduction Nam and Li, 2002). The BR signal transduction pathway ultimately controls the phosphorylation status of the transcription factors BZR1 and BZR2/BES1 in the nucleus (Kim et al., 2009) and thereby regulates the expression of more than 700 genes in Arabidopsis (Arabidopsis thaliana; Goda et al., 2002;Mü ssig et al., 2002;Vert et al., 2005). Many specific components of the signal transduction pathway have been identified and have recently been reviewed (Kim and Wang, 2010;Tang et al., 2010).BRs are now considered essential chemical signals and plant hormones, and accordingly, manipulation of endogenous BR content or BR signaling has a profound effect on plant growth. For example, overexpression of DWARF4, which encodes an enzyme that catalyzes a rate-limiting step in BR biosynthesis, enhances plant growth and seed yield in Arabidopsis (Choe et al., 2001), while down-regulation of BR biosynthesis...

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The balance of reproducibility, sensitivity, and specificity of lists of differentially expressed genes in microarray studies

Cited by 212 publications

References 54 publications

Improved RNA preservation for immunolabeling and laser microdissection

Improved RNA preservation for immunolabeling and laser microdissection

Paneth cell marker expression in intestinal villi and colon crypts characterizes dietary induced risk for mouse sporadic intestinal cancer

Enhancing Arabidopsis Leaf Growth by Engineering the BRASSINOSTEROID INSENSITIVE1 Receptor Kinase

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