Simple SummaryEndogenous reference genes in diagnostic specimens are used to monitor sample quality in a quantitative polymerase chain reaction (qPCR), i.e., serve the role of internal sample controls (ISC). However, there is little information on the consistency of ISC expression among specimen types or the interpretation of ISC results. Therefore, the aim of this study was to evaluate the expression of a porcine-specific ISC in serum, oral fluid, and fecal specimens collected from pigs of known porcine reproductive and respiratory syndrome virus (PRRSV) infection status and tested using a commercial PRRSV reverse transcription-qPCR. The ISC was detected in 100% of the specimens tested and was not affected by PRRSV infection status of the pigs, but ISC concentration varied between specimen types. Thus, reference limits were established for each specimen to provide guidelines for ISC interpretation. Overall, the ISC evaluated herein can be used to accurately monitor sample quality in swine specimens tested for PRRSV. In particular, failure to detect the ISC indicates an irregularity with the sample or the testing procedures.AbstractEndogenous reference genes are used in gene-expression studies to “normalize” the results and, increasingly, as internal sample controls (ISC) in diagnostic quantitative polymerase chain reaction (qPCR). Three studies were conducted to evaluate the performance of a porcine-specific ISC in a commercial porcine reproductive and respiratory syndrome virus (PRRSV) reverse transcription-qPCR. Study 1 evaluated the species specificity of the ISC by testing serum from seven non-porcine domestic species (n= 34). In Study 2, the constancy of ISC detection over time (≥42 days) was assessed in oral fluid (n= 130), serum (n= 215), and feces (n= 132) collected from individual pigs of known PRRSV status. In Study 3, serum (n= 150), oral fluid (n= 150), and fecal samples (n= 75 feces, 75 fecal swabs) from commercial herds were used to establish ISC reference limits. Study 1 showed that the ISC was porcine-specific, i.e., all samples from non-porcine species were ISC negative (n= 34). In Study 2, the ISC was detected in all oral fluid, serum, and fecal samples, but differed in concentration between specimens (p< 0.05; mixed-effects regression model). The results of Study 3 were used to establish ISC reference limits for the 5th, 2.5th and 1.25th percentiles. Overall, the ISC response was consistent to the point that failure in detection is sufficient justification for re-testing and/or re-sampling.