In this review, we discuss the development pipeline for transcriptional biomarkers in molecular diagnostics and stress the importance of a reliable gene transcript quantification strategy. Hence, a further focus is put on the MIQE guidelines and how to adapt them for biomarker discovery, from signature validation up to routine diagnostic applications. First, the advantages and pitfalls of the holistic RNA sequencing for biomarker development will be described to establish a candidate biomarker signature. Sequentially, the RT-qPCR confirmation process will be discussed to validate the discovered biomarker signature. Examples for the successful application of RT-qPCR as a fast and reproducible quantification method in routinemolecular diagnostics are provided. Based on the MIQE guidelines, the importance of “key steps” in RT-qPCR is accurately described, e.g., reverse transcription, proper reference gene selection and, finally, the application of automated RT-qPCR data analysis software. In conclusion, RT-qPCR proves to be a valuable tool in the establishment of a disease-specific transcriptional biomarker signature and will have a great future in molecular diagnostics or personalized medicine.

Introduction: MicroRNAs have a significant role in the regulation of the transcriptome. Several miRNAs have been proposed as potential biomarkers in different malignancies. However, contradictory results have been reported on the capability of miRNA biomarkers in cancer detection. The human biological clock involves molecular mechanisms that regulate several genes over time. Therefore, the sampling time becomes one of the significant factors in gene expression studies.Method: In the present study, we have tried to find miRNAs with minimum fluctuation in expression levels at different time points that could be more accurate candidates as diagnostic biomarkers. The small RNA-seq raw data of ten healthy individuals across nine-time points were analyzed to identify miRNAs with stable expression.Results: We have found five oscillation patterns. The stable miRNAs were investigated in 779 small-RNA-seq datasets of eleven cancer types. All miRNAs with the highest differential expression were selected for further analysis. The selected miRNAs were explored for functional pathways. The predominantly enriched pathways were miRNA in cancer and the P53-signaling pathway. Finally, we have found seven miRNAs, including miR-142-3p, miR-199a-5p, miR-223-5p, let-7d-5p, miR-148b-3p, miR-340-5p, and miR-421. These miRNAs showed minimum fluctuation in healthy blood and were dysregulated in the blood of eleven cancer types. Conclusion: We have found a signature of seven stable miRNAs which dysregulate in several cancer types and may serve as potential pan-cancer biomarkers.