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.

Fig. 1

Generalized workflow for primer validation. Primers first met PCR efficiency criteria (80–120 %). Secondary evaluation of target specificity used high resolution melt temperature analysis to confirm the presence of a single melt peak. Final confirmation of primer specificity utilized electrophoresis through agarose to ensure only one product was produced, validating the primer pair, and establishing the expected T _m for each corresponding amplimer. Approximately 40 % of all amplimers were sequenced with 100 % confirmation of their identities

Fig. 2

Secondary primer pair validation was conducted by melt temperature (T _m) analysis to confirm target specificity. All reactions were carried out as three, 10-fold dilutions analyzed in duplicate. a A single melt peak indicated production of one product and a passed secondary evaluation. b Multiple melt peaks were indicative of off-target primer binding or primer-dimer formation, resulting in multiple amplification products. The lower panel primer pair did not pass validation and was re-designed. c An example of a primer pair that produced a primer-dimer in the no template control well. This primer pair passed validation as the T _m of the primer-dimer was ≥5 °C from the expected T _m. All panels: the dotted line indicates the baseline threshold

Fig. 3

Comparisons between MG 2300 and MG 2341 showed significant transcription level changes in 42 % of genes evaluated. A plot comparing MG 2300 to MG 2341 showed that significant up-regulation was detected for 13 genes (upper right quadrant). Additionally, 27 genes were significantly down-regulated (upper left quadrant). The gray rectangle indicates a 3-fold up- or down-regulation. Approximately, 58 % of observed gene transcription levels were found to fall within this range and were considered unchanged. Selected genes were evaluated by qPCR, and both their magnitude and direction of change were in agreement with the data obtained from the array. The dotted line indicates a P value of 0.05

Fig. 4

Initial validation of the gpArray using guinea pig splenocytes treated with medium only for 24 h (unstimulated) or PMA/ionomycin (stimulated). Both (a) and (b). The solid line indicates baseline expression; the dotted lines indicated 3-fold up or down expression differences. a Replicates of unstimulated splenocytes showed no differences validating the reproducibility of the array and establishing the level of biological and technical noise. b When stimulated splenocytes were compared to unstimulated samples, 16 % of genes were found to be up-regulated, 60 % were down-regulated, and the remaining genes showed no change. Both (c) and (d). The dotted line indicates P value of 0.05. The light gray rectangle indicates a 3-fold up or down change in expression levels. Gene transcription levels within this range were considered unchanged. c Statistical significance of expression level differences from stimulated splenocytes compared to unstimulated; 15 genes were significantly up-regulated (upper right quadrant) and 55 were significantly down-regulated (upper left quadrant). d qPCR results were concordant for direction of change and P value for 54 % of genes evaluated (indicated by Black filled star). Genes that were discordant with respect to direction, P value or both were found within a 5-fold up- or down-regulation and are denoted by (×). The dark gray rectangles indicate ±5-fold change in expression levels with all genes that showed >5-fold change corresponding to 100 % concordance with array data as confirmed by qPCR