To obtain reliable data using RT-qPCR, gene expression levels must be normalized using internal controls within each sample. The use of one or more 4-IBP reference genes can correct biases caused by variations in the complementary DNA input or the efficiency of reverse transcription or amplification. Ideally, reference genes should be stably expressed or at least vary only slightly in expression in all tissues or cells under the conditions of the experiment. The suitability of reference genes has been evaluated in some human musculoskeletal diseases, such as shoulder instability, osteoarthritic articular cartilage, human lumbar vertebral endplate with modic changes and skeletal muscle with chronic degenerative changes. Using a semi-quantitative approach, Lo et al. described that they evaluated several widely accepted housekeeping genes and observed that GAPDH mRNA levels are constant in dense connective tissues at different times in both normal and injured/healing tissue. Although the gene stability data were not provided, GAPDH and ACTB have been used as a reference genes in the study of mRNA regulation in human rotator cuff tear. Reference genes have been described for RT-qPCR studies in several diseases and tissues, and our group recently identified the most stable reference genes in the glenohumeral capsule of patients with and without shoulder instability. To the best of our 2-PMDQ knowledge, no prior study has aimed to identify suitable reference genes for gene expression analyses by quantitative approaches in human tendons. In the present study, we used 4 software packages to evaluate the stability of reference gene expression. As each software package implements distinct algorithms, different results can be expected. Therefore, it is important to use more than one software package to identify the most suitable reference genes among a set of candidates. Although the 4 software packages differed in their rankings of reference gene stability as well as in the identity of the most suitable pair, at least two programs produced results that agreed for almost all the analyses. Our results demonstrate that the use of 4 statistical tools aids in the identification of the best reference genes. All the reference genes in this study presented an Mvalue less than the geNorm threshold of 1.5 recognized as stable under the different experimental conditions tested. However, 18S and B2M presented high SD of Crt in the analysis involving all samples according to BestKeeper software. Therefore, these 2 reference genes should not be used in analysis involving different types and conditions of tendon of rotator cuff. In the different groups of analyses, HPRT1 appeared to be the most suitable gene overall; however, it is increasingly clear that in most situations, a single reference gene is not sufficiently stable.