Development and Utilization of a Custom PCR Array Workflow

Analysis of Gene Expression in Mycoplasma genitalium and Guinea Pig (Cavia porcellus)

Ronald L. Veselenak, Aaron L. Miller, Gregg Milligan, Nigel Bourne, Richard Pyles

Research output: Contribution to journalArticle

7 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)172-183
Number of pages12
JournalMolecular Biotechnology
Volume57
Issue number2
DOIs
StatePublished - 2014

Fingerprint

Mycoplasma genitalium
Workflow
Gene expression
Guinea Pigs
Gene Expression
Polymerase Chain Reaction
Microarrays
RNA
Biological Phenomena
Rats
Costs
Costs and Cost Analysis
Assays
Bacteria
Genes
Gene Expression Profiling
Messenger RNA
Genome

Keywords

  • Cavia porcellus
  • Gene expression
  • Guinea pig
  • Immune response
  • Mycoplasma genitalium
  • PCR array
  • Transcription

ASJC Scopus subject areas

  • Biochemistry
  • Biotechnology
  • Molecular Biology
  • Bioengineering
  • Applied Microbiology and Biotechnology

Cite this

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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.",
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AU - Bourne, Nigel

AU - Pyles, Richard

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