Tissue-specific expression pattern of bovine prion gene: quantification using real-time RT-PCR

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Abstract

In recent studies PrP mRNA was determined mostly by in situ hybridisation or Northern Blot analysis—methods not suitable for absolute quantification of mRNA copy numbers. Herein we report on bovine prion mRNA quantification using calibrated highly sensitive externally standardized real-time RT-PCR with LightCycler instrument. Total RNA was isolated from nine different regions of the CNS and seven peripheral organs. PrPc mRNA copy numbers could be determined in all tissues under study. In approval with prior studies high mRNA level was found in Neocortex and Cerebellum. Lymphatic organs showed at least as high expression levels of prion mRNA as overall brain. Lowest expression was detected in kidney. Results of our study provide insight into the involvement of different organs in pathogenesis with respect to prion mRNA expression. LightCycler technology is currently considered the most precise method for nucleic acid quantification and showed to be powerful tool for further studies on prion diseases pathogenesis.

Introduction

Cellular prion protein (PrPc) [1] a glycosylphosphatidylinositol (GPI) anchored glycoprotein [2] expressed in numerous cell types [3] and tissues is suspected to be involved in the pathogenesis of prion diseases [4], [5]. These neurodegenerative disorders are described in many species such as cattle (BSE), sheep (Scrapie), mink (TME), cats, (FSE) and also in humans (CJD) (for overview see 5). Alterations are histopathologically characterised by accumulation of pathogenic prion protein (PrPsc) isoform. During disease progression PrPc serves as a substrate molecule for PrPsc that acts as a template [4], [5], [6]. Due to direct interaction between these two types of molecules PrPc undergoes autocatalytic conformational changes and turns PrPsc. Unlike PrPc that has a more α-helical content, PrPsc mainly shows β-sheeted structure [7], [8]. As no other pathogens or nucleic acids seem to be involved in this process, it is called the ‘protein-only hypothesis’ [9].

Pathological alterations are mostly related to the central nervous system (CNS), but some early studies indicated that in pre-clinical stages of disease progression peripheral organs might play a crucial role [10], [11], [12] in pathogenesis. In this regard lymphoid organs are already of long-term high interest [13], [14].

Expression of prion gene in neuronal and non-neuronal tissues has to be taken into special consideration for a better understanding of its role in organism as well as in prion disease pathogenesis and for consumption risk assessment. Spread of PrPsc from peripheral organs to the CNS is poorly understood to date. Nevertheless it becomes more apparent, that cells of the immune system play an important role in PrPsc accumulation and distribution [15], [16]. Amount of PrPc mRNA in these cells and subsequent translation product abundance probably play a role in disease initiation and progression. It is likely that cells with higher expression of PrPc pose higher risk of conversion to and accumulation of PrPsc.

Real-time RT-PCR using SYBR Green I technology [17] provides an excellent and highly sensitive method for absolute quantification of mRNA expression. Using an external calibration curve based on plasmid DNA the quantification model showed higher sensitivity, exhibited a larger quantification range, had a higher reproducibility, than models using recombinant RNA or diluted PCR product as calibration curve [18].

Getting insight into prion gene expression in tissues and organs possibly under various treatments is an essential starting point for further study of protein conversion and PrPsc accumulation. Tissue-specific expression pattern determined with high reproducibility and accuracy is also essential for understanding poorly explained natural role of prions in organism. Herein we show results concerning prion mRNA expression in CNS and peripheral organs as well as we test suitability of above-mentioned method.

Section snippets

Material and methods

Three healthy male Holstein-Frisian calves at the age of six month and three healthy adult ‘Brown Swiss’ cows were selected for tissue material sampling. Animals were slaughtered using ordinary procedure according to EU's established hygienic policy at the Bayerisch Landesanstalt für Tierzucht, Grub. Following organs were sampled: Neocortex, Cerebellum, Thalamus, Hypothalamus, Pituitary gland, Medulla oblongata, Pars cervicalis, Pars thoracalis and Pars lumbalis, this all with respect to CNS.

Results

Sensitivity of the LightCycler RT-PCR was evaluated using different starting amounts of mRNA and standard curve. SYBR Green I fluorescence determination at the elevated temperature 83 °C resulted in a reliable and sensitive cDNA quantification assay with high linearity (Pearson correlation coefficient=0.99) over five orders of magnitude from 2×103 to 2×107 recombinant standard DNA start molecules (Fig. 1).

To verify real-time RT-PCR products derived either from plasmid or tissue total RNA a

Discussion and conclusions

Intuitive accord of our results with previously published data shows that real-time RT-PCR using LightCycler is reliable and powerful instrument to study pathogenesis of prion disease on mRNA level. Nevertheless, it makes these studies comparable and reproducible. It yields valued outputs as to levels of prion mRNA in different tissues. The reverse transcription reaction is the most significant cause of error. From our and other's experiences. We consider its efficiency between 30–70%,

Acknowledgements

We thank the Bayerische Landesanstalt für Tierzucht at Grub for excellent technical support during the slaughtering and tissue sampling process.

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