Selection of Biomarkers from Differentially Expressed Genes in Leukocytes of Buffalos Treated with Recombinant Bovine Somatotropin: The Importance of Sample Size for Reliable Discriminating Systems

The research on biomarkers to detect livestock treated with recombinant bovine somatotropin (rbST) is still an open issue. In fact, beyond undertaking confirmation methods, there is the need to develop simple and inexpensive screening tests. In this direction, some proposals have been forwarded, mostly involving the measurement of circulating molecules, whereas the possibility of using biomarkers related to gene expression is a field under investigation. The present study was carried out on sixteen buffalos, eight of which treated with rbST. Blood samples were collected six times during the treatment to investigate on the presence of differentially expressed genes in leukocytes. Analysis with the microarray technique was performed on two sampling moments, in order to obtain a first selection of genes. Further analysis was carried out by real time RT-PCR, in order to create a discriminating linear system. A study on the variation of the error related to the number of samples included in statistics was also performed. Results showed that, including an increasing number of samples to build the discriminating algorithm, the b-error grows and tends to stabilize on 6.5%. This study clearly shows the paramount importance of including a proper number of samples to obtain reliable algorithms

Analisi di espressione genica in leucociti di bufale trattate con somatotropina bovina ricombinante: ricerca di biomarcatori.

La Somatotropina bovina ricombinante (rbST) è un ormone proteico di sintesi prodotto da alcune grandi multinazionali farmaceutiche e commercializzato in ambito zootecnico allo scopo di incrementare la resa lattea degli animali in produzione. Sebbene in alcune parti del mondo il suo utilizzo sia stato autorizzato, l’Unione Europea ne ha bandito il commercio e l’impiego all’ interno dei suoi stati membri (Decisione del Consiglio 1999/879/CE). Il recente ritrovamento di farmaci iniettabili a lento rilascio a base di rbST in alcuni allevamenti di bufale italiani ha ulteriormente confermato la necessità di disporre di strumenti analitici in grado di svelare eventuali trattamenti illeciti. Ad oggi, non esistono test di screening validati con questo proposito. Questo lavoro di tesi si inserisce in un progetto che si propone di analizzare la variazione dell’espressione genica in leucociti di bufale alle quali è stata somministrata rbST, con lo scopo di ricercare biomarcatori come indice del trattamento ormonale. L’RNA estratto dalle cellule bianche di 8 animali trattati e 8 controlli, in 6 momenti di campionamento, all’interno di 3 cicli di somministrazione bisettimanale del farmaco a lento rilascio a base di rbST, è stato analizzato mediante microarray, al fine di selezionare i geni differenzialmente espressi in un contesto di circa 44.000 geni totali. In un secondo momento l’analisi è stata approfondita tramite Real Time PCR. I risultati sono stati sottoposti ad analisi discriminante lineare, allo scopo di individuare le variabili (geni) con maggior potere discriminante, utilizzate, a loro volta, per elaborare un sistema lineare classificante. La percentuale di falsi negativi ottenuta è stata circa 6.5%. Anche se la Decisione 2002/657/CE prevede che un test di screening debba essere caratterizzato da un tasso di falsi negativi inferiore al 5%, il test oggetto di questo studio potrebbe essere ulteriormente perfezionato aumentando la popolazione statistica e selezionando algoritmi che privilegino la sensibilità rispetto alla specificità. The recombinant bovine somatotropin (rbST) is a synthetic peptide hormone produced and commercialized by several large international pharmaceutical companies in order to increase milk yield in livestock. Although its use has been authorized in some countries worldwide, the European Union has banned its trade and use within its member States (Council Decision 1999/879 / EC). However, the use in some Italian buffaloes herds of slow- release injectable drugs containing rbST has been recently discovered, further confirming the need for analytical tools capable of revealing any illegal treatment. To date, no validated screening tests exist. This thesis is part of a project that aims to analyze the variation of gene expression in leukocytes of buffaloes treated with rbST, in order to seek hormonal treatment biomarkers. RNA extracted from white cells of 8 treated animals and 8 controls, in 6 sampling times, within 3 cycles of biweekly administration of the rbST slow- release drug, was analyzed by microarray, in order to select differentially expressed genes out of a total of about 44,000 genes. In a second phase, the analysis was refined by Real Time PCR. The results were submitted to linear discriminant analysis, in order to identify the variables with the greatest discriminating power. The identified variables were then used to develop a linear system for classification. A percentage of false negatives of approximately 6.5% was obtained. The classifying algorithm may be further improved to reach a false negative rate of less than 5% as required by the decision 2002/657/EC. This could be done by increasing the statistical population and by selecting algorithms that favor sensitivity rather than specificity