Since transcriptome analysis provides genome-wide sequence and gene expression information, transcript reconstruction using RNA-Seq sequence reads has become popular during recent years. For non-model organism, as distinct from the reference genome-based mapping, sequence reads are processed via de novo transcriptome assembly approaches to produce large numbers of contigs corresponding to coding or non-coding, but expressed, part of genome. In spite of immense potential of RNA-Seq-based methods, particularly in recovering full-length transcripts and spliced isoforms from short-reads, the accurate results can be only obtained by the procedures to be taken in a step-by-step manner. In this chapter, we aim to provide an overview of the state-of-the-art methods including (i) quality check and pre-processing of raw reads, (ii) the pros and cons of de novo transcriptome assemblers, (iii) generating non-redundant transcript data, (iv) current quality assessment tools for de novo transcriptome assemblies, (v) approaches for transcript abundance and diferential expression estimations and inally (vi) further mining of transcriptomic data for particular biological questions. Our intention is to provide an overview and practical guidance for choosing the appropriate approaches to best meet the needs of researchers in this area and also outline the strategies to improve on-going projects.

Arsenic is a broad-spectrum environmental contaminant with mutagenic, teratogenic and carcinogenic effects. Due to its widespread distribution in nature, drinking water is the most common source of arsenic exposure for the general population. In this study, we aimed to determine the effect of sodium arsenite on the viability and expression profile of steroidogenic genes in TM3 Leydig cells, responsible for testicular steroidogenesis. The TM3 Leydig cells were treated with sodium arsenic (384,8 µM or 7,6 mM) for 24 hours with LH (Luteinizing hormone) stimulation. The MTT assay was used for measuring cell viability, the expression level of key genes of the steroidogenesis was evaluated using RT-qPCR.The MTT assay showed that cell viability was decreased dose-dependently. RT-qPCR demonstrated that the expression level of CYP11A1, CYP17A1 were decreased as compared to the untreated control while StAR gene expression was found to be surprisingly high in the cell exposed to high-dose arsenic (p<0.05). The expression profile of two dehydrogenase; 17β-HSD and 3β-HSD was significantly reduced in high dose arsenic treated-group (p<0.05); but however, no statistical significance was found in low-dose. The RT-qPCR also showed that the expression SF-1 (Steroidogenic factor-1), a key regulator of adrenal and reproductive development, was significantly decreased in both low-dose and high-dose (p<0.05). Arsenic toxicity in Leydig cell leads to cell viability loss and cause a perturbation in key steroidogenic genes, reflecting the possible role of arsenic in infertility and male reproductive system disorders.

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