title: Development and utilization of a custom PCR array workflow: analysis of gene expression in mycoplasma genitalium and guinea pig (Cavia porcellus).
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.
authors.affiliation: Department of Microbiology and Immunology, University of Texas Medical Branch, 301 University Blvd, Galveston, TX, 77555-0436, USA.
authors.forename: Ronald L
authors.initials: RL
authors.lastname: Veselenak
authors.forename: Aaron L
authors.initials: AL
authors.lastname: Miller
authors.forename: Gregg N
authors.initials: GN
authors.lastname: Milligan
authors.forename: Nigel
authors.initials: N
authors.lastname: Bourne
authors.forename: Richard B
authors.initials: RB
authors.lastname: Pyles
mesh_terms: D000818:Animals; D015870:Gene Expression; D020869:Gene Expression Profiling; D006168:Guinea Pigs; D045704:Mycoplasma genitalium / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*