Abstract
Transcriptome analysis is a powerful tool for evaluating molecular pathways central to maturation of specific biological processes and disease states. Recently, PCR-based arrays have supplemented microarray and RNA-seq methodologies for studying changes in gene expression levels. PCR arrays are a more cost efficient alternative, however commercially available assemblies are generally limited to only a few more widely researched species (e.g., rat, human, and mouse). Consequently, the investigation of emerging or under-studied species is hindered until such assays are created. To address this need, we present data documenting the success of a developed workflow with enhanced potential to create and validate novel RT-PCR arrays for underrepresented species with whole or partial genome annotation. Utilizing this enhanced workflow, we have achieved a success rate of 80 % for first-round designs for over 400 primer pairs. Of these, ~160 distinct targets were sequence confirmed. Proof of concept studies using two unique arrays, one targeting the pathogenic bacterium Mycoplasma genitalium and the other specific for the guinea pig (Cavia porcellus), allowed us to identify significant (P < 0.05) changes in mRNA expression validated by subsequent qPCR. This flexible and adaptable platform provides a valuable and cost-effective alternative for gene expression analysis.Electronic supplementary materialThe online version of this article (doi:10.1007/s12033-014-9813-6) contains supplementary material, which is available to authorized users.
Highlights
For many novel emerging or under-studied organisms, the lack of assays to quantify and characterize proteins and phenotype necessitates the evaluation of nucleic acids
We present data documenting the success of a developed workflow with enhanced potential to create and validate novel reverse transcription PCR (RT-PCR) arrays for underrepresented species with whole or partial genome annotation
We present the successful development and validation of two disparate arrays targeting the bacterial pathogen Mycoplasma genitalium (MG) and the commonly utilized guinea pig (Cavia porcellus), both of which currently lack commercially available assays for transcriptome analysis
Summary
For many novel emerging or under-studied organisms, the lack of assays to quantify and characterize proteins and phenotype necessitates the evaluation of nucleic acids. While partial or complete genomic sequence may be available for many of these organisms, the expertise and cost required for de novo sequencing or to develop assays for measuring transcription level changes can be a significant deterrent to the development of custom in-house reagents. The lack of demand for niche products to address under-studied or emerging organisms further diminishes the likelihood of commercial production of reagents. Transcriptome analysis by either microarray or RNA-seq is currently widely utilized in a discovery role for large-scale gene expression studies [3] and transcript-level analyses made possible by both technologies have revolutionized molecular biology [2, 4]
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