PMC:3886832 / 4210-12254 JSONTXT

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Experimental Methodologies\nThe state of a cell can be understood by the genes that are transcribed at a given time. Gene expression profiling studies (i.e., transcriptomics) are tremendously useful in providing a snapshot of what mRNA transcripts are made in the cell. Both microarray and NGS technology are widely used to understand the expression of genes under varying conditions (disease, developmental time points, following stress, etc.). A PubMed search with either the key word “microarray” or “next-generation-sequencing” showed that the reference of these technologies has increased tremendously in the last decade, from only a few papers in the late 1990s to over 7,000 publications in 2012. NGS technology is a relatively newer subset of these publications and has increased considerably since 2008 (Figure 1). Each microarray or NGS study can result in huge datasets of hundreds to thousands of differentially expressed transcripts, which has led to an explosion of new data. As a result, looking for cellular pathways specific to hair cell regeneration can be like looking for a hair cell in a haystack, so to speak. Fortunately, bioinformatics tools are being developed alongside molecular tools to help filter all the rapidly growing data and assist in finding the most important cellular networks of interest.\nFigure 1 Graph indicating the number of articles in PubMed including one of the two keywords “microarray” or “next-generation-sequencing” in their articles. Microarray technology evolved from the traditional dot blot technique that was used for parallel screening of small numbers of genes, and it works by the same principle of hybridization between the target DNA and the probe [31,32]. Complementary DNA (cDNA) and oligonucleotide arrays are two miniaturized forms of dot blot that can be used to identify thousands of genes at the same time. In the cDNA array, a control and an experimental sample are fluorescently labeled separately using different dyes and applied on the same array to determine the relative intensities of genes printed on the chip. Later, with the development of oligonucleotide arrays, the need for adding a control sample along with the experimental sample was overcome [32]. This array technology has been widely used in many different organisms, tissues and in vitro culture studies relevant to the biology of the inner ear (Table 1). TaqMan low density arrays are another type of array that are based on the real-time quantitative reverse transcription-PCR (QRT-PCR) method to study more focused gene expression patterns in tissues. This method is more sensitive than gene chips and offers higher throughput than standard QRT-PCR [33]. Serial analysis of gene expression (SAGE), another technique used to identify genes that are differentially expressed, is based on unique tag sequences that are sufficient to identify different transcripts [34]. Later, the advent of NGS technology completely changed the way of analyzing gene expression data. Unlike microarray, this technology works by identifying genes by sequencing fragmented short reads. Both SAGE and NGS eliminate the need for prior knowledge of probes to scan for genes and, thus, have the potential to identify alternative splice variants and novel transcripts, with greater sensitivity for the transcripts that are expressed at low levels. NGS comes in different platforms that vary in sequencing chemistry and can be applied to study questions at different biological levels relating to the transcriptome or epigenome [35,36]. While the transcriptome is the set of transcribed RNAs that are found in a tissue, the epigenome examines regulation of gene expression via DNA cytosine methylation or deamination, histone protein modifications, such as acetylation, genomic imprinting and RNA editing. Understanding of the role of epigenetics in hair cell development or regeneration is still in its infancy, but early studies have been reviewed elsewhere [37].\nmicroarrays-02-00186-t001_Table 1 Table 1 Representative studies examining gene expression in the inner ear during development, in different cell/tissue types and following trauma. Both microarray and NGS have been applied to understand processes related to hair cell regeneration in the inner ear of vertebrates. Three basic experimental paradigms have been utilized. (1) Since the process of regeneration and development may have redundant pathways, understanding temporal shifts in gene expression patterns during the normal development of the auditory system and the production of new hair cells may provide clues to important cellular pathways used for hair cell regeneration. (2) Another clue to processes involved in hair cell regeneration is gene expression patterns between auditory sensory tissues that differ in their ability to regenerate. The mature mammalian sensory epithelium of the cochlea do not form new hair cells post-development or following hair cell damage or loss, but maintain mitotic quiescence [45,46]. In contrast, the mammalian vestibular epithelium exhibits more plasticity, and damage to the utricle has led to supporting cell proliferation [47] and even some cells differentiating into cells with hair cell-like characteristics [48,49]. Thus, gene profiling studies contrasting differences between quiescent and regenerating tissues may bring to light pathways that are activated during hair cell regeneration. (3) Lastly, the most direct method of examining genes and pathways important during hair cell regeneration is to damage the inner ear with either ototoxic chemicals, acoustical overstimulation or mechanical ablation and, then, analyze shifts in gene expression at specified time points during the recovery process. Some representative studies that have used these three experimental paradigms during the last decade are summarized in Table 1. While much of the focus has been on gene expression patterns (transcriptomics) as a means of understanding important cellular processes involved in hair cell regeneration, regulation of these processes can also occur at the post-transcriptional, translational and post-translational levels. In fact, mRNA levels often do not correspond well with protein expression levels, because protein abundance is dynamic [50]. For example, some proteins are constitutively expressed, and their functionality is mediated by phosphorylation or other post-translational events. In addition, mRNA levels cannot predict post-translational modifications or which splice variants are translated. \nAs a result, there has been a surge in research on post-transcriptional control (primarily examining microRNAs) and proteomics in the inner ear. The study of proteomics started from 2D gel electrophoresis in which the proteins are first separated in the first dimension by their isoelectric point and, then, in the second dimension by their mass. The separated proteins can then be cut out, in-gel trypsin digested and analyzed using mass spectrometry. Using 2D-difference gel electrophoresis (2D-DIGE), different experimental samples can be labeled separately and mixed together before isoelectric point separation, similar to sample preparation for microarrays [31,51]. Large-scale protein analysis can be studied by using protein arrays that contain antibodies attached to the chip to detect its specific antigens [52]. Liquid chromatography in tandem with mass spectrometry (LC-MS) can be used to identify large numbers of proteins, and it can detect low molecular proteins that are less than 5,000 KDa [31]. The post-translational modification of proteins can be identified from such proteomic techniques, but not from gene microarrays. 2D-DIGE and LC-MS methods have been used to study the inner ear proteome [53,54,55,56,57]. Together, large-scale transcriptome, microRNA and proteome studies have provided and continue to provide copious amounts of data to mine in order to understand the process of hair cell regeneration in the inner ear at both the transcript and protein level.\n"}