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 language | English (US) |
|---|---|
| Pages (from-to) | 172-183 |
| Number of pages | 12 |
| Journal | Molecular Biotechnology |
| Volume | 57 |
| Issue number | 2 |
| DOIs | |
| State | Published - 2014 |
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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
Development and Utilization of a Custom PCR Array Workflow : Analysis of Gene Expression in Mycoplasma genitalium and Guinea Pig (Cavia porcellus). / Veselenak, Ronald L.; Miller, Aaron L.; Milligan, Gregg; Bourne, Nigel; Pyles, Richard.
In: Molecular Biotechnology, Vol. 57, No. 2, 2014, p. 172-183.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Development and Utilization of a Custom PCR Array Workflow
T2 - Analysis of Gene Expression in Mycoplasma genitalium and Guinea Pig (Cavia porcellus)
AU - Veselenak, Ronald L.
AU - Miller, Aaron L.
AU - Milligan, Gregg
AU - Bourne, Nigel
AU - Pyles, Richard
PY - 2014
Y1 - 2014
N2 - 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.
AB - 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.
KW - Cavia porcellus
KW - Gene expression
KW - Guinea pig
KW - Immune response
KW - Mycoplasma genitalium
KW - PCR array
KW - Transcription
UR - http://www.scopus.com/inward/record.url?scp=84921416157&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84921416157&partnerID=8YFLogxK
U2 - 10.1007/s12033-014-9813-6
DO - 10.1007/s12033-014-9813-6
M3 - Article
C2 - 25358686
AN - SCOPUS:84921416157
VL - 57
SP - 172
EP - 183
JO - Molecular Biotechnology
JF - Molecular Biotechnology
SN - 1073-6085
IS - 2
ER -