Transcriptomics

Biomarkers able to predict response and toxicity upon regorafenib therapy for colorectal cancer (CRC) are critical for treatment choice, particularly relevant in fragile patients. Here, we validated for the first time 18 distinct microRNAs (miRNAs) detected in serum and primary tumor samples, three germline single-nucleotide polymorphisms (SNPs) in vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor (VEGFR) genes, and low levels of Notch 1 expression in the primary tumor as predictive biomarkers of different features. Specifically, these markers were associated with a favorable response to treatment, disease stage, and relapse, as well as the appearance of asthenia. Therefore, these markers can be potentially useful biomarkers for patient stratification and for providing a more personalized and effective therapeutic strategy in fragile patients, while limiting the appearance of adverse effects.

microRNAs (miRNA) expression in colorectal (CR) primary tumours can facilitate a more precise molecular characterization. We identified and validated a miRNA profile associated with clinical and histopathological features that might be useful for patient stratification. In situ hybridization array using paraffin-embedded biopsies of CR primary tumours were used to screen 1436 miRNAs. 17 miRNAs were selected for validation by quantitative reverse transcription polymerase chain reaction (qRT-PCR) (n = 192) and were further correlated with clinical and histopathological data. We demonstrated that miRNAs associated to Colorectal Cancer (CRC) diagnosis age (over 50s and 60s) included miR-1-3p, miR-23b-3p, miR-27b-3p, miR-143-3p, miR-145-5p and miR-193b-5p. miR-23b-3p and miR-24-3p discriminated between Lynch Syndrome and sporadic CRC. miR-10a-5p, miR-20a-5p, miR-642b and Let-7a-5p were associated to stroma abundance. miR-642b and Let-7a-5p were associated with to peritumoral inflammation abundance. miR-1-3p, miR-143-3p and miR-145-5p correlated with mucinous component. miR-326 correlated with tumour location (right or left sided). miR-1-3p associated with tumour grade. miR-20a-5p, miR-193b-5p, miR-320a, miR-326 and miR-642b-3p associated to tumour stage and progression. Remarkably, we also demonstrated that miR-1-3p and miR-326 expression significantly associated with patient overall survival (OS). Hierarchical clustering and bioinformatics analysis indicated that selected miRNAs could re-classify the patients and work cooperatively, modulating common target genes involved in colorectal cancer key signalling pathways. In conclusion, molecular characterization of CR primary tumours based on miRNAs could lead to more accurate patient reclassification and may be useful for efficient patient management.

Despite promising findings, quantitative PCR (qPCR)-based tests for RNA quantification have experienced serious limitations in their clinical application. The noticeable lack of technical standardization remains a huge obstacle in the translation of qPCR-based tests. The incorporation of qPCR-based tests into the clinic will benefit from guidelines for clinical research assay validation. This will ultimately impact the clinical management of the patient, including diagnosis, prognosis, prediction, monitoring of the therapeutic response, and evaluation of toxicity. However, clear assay validation protocols for biomarker investigation in clinical trials using molecular assays are currently lacking. Here, we will focus on the necessary steps, including sample acquisition, processing and storage, RNA purification, target selection, assay design, and experimental design, that need to be taken toward the appropriate validation of qRT-PCR assays in clinical research. These recommendations can fill the gap between research use only (RUO) and in vitro diagnostics (IVD). Our contribution provides a tool for basic and clinical research for the development of validated assays in the intermediate steps of biomarker research. These guidelines are based on the current understanding and consensus within the EU-CardioRNA COST Action consortium (www.cardiorna.eu). Their applicability encompasses all clinical areas.

Protocol for miRNAs extraction from serum/plasma samples using miRNeasy Serum/plasma advanced QIAGEN kit (Reference number: 217204) by RT-PCR in RNA from different samples, including appropriate controls and normalizers for further RT-PCR data analysis

Protocol detailing the essential steps for preparing plasma samples in liquid biopsy studies, with a specific focus on miRNA analysis. Proper plasma preparation is crucial to ensure accurate and reliable results in miRNA studies, which hold significant potential for non-invasive disease diagnostics and monitoring.

Protocol detailing the essential steps for preparing serum samples in liquid biopsy studies, with a specific focus on miRNA analysis. Proper plasma preparation is crucial to ensure accurate and reliable results in miRNA studies, which hold significant potential for non-invasive disease diagnostics and monitoring.

Protocol for stranded sequencing mRNA extracted from human serum

Protocol for preparation and sequencing  of microRNA extracted from (blood) cells

Protocol for First-Strand cDNA Synthesis: miRNA Retrotranscription (RT) from serum or plasma using Qiagen kit (reference number 339340)

Protocol for miRNAs amplification by qPCR using miRCURY LNA miRNome PCR Panels or Serum/Plasma Focus PCR Panels Qiagen kit (Reference number 339325)

The qPCR has been introduced in clinical and biomedical research for over 10 years from now. Its use in trials and diagnostics is continuously increasing. Due to this heavy use, the question of relyability and relevance of qPCR results has to be asked. This review proposes a documented and evidence based answer to this question, thanks to the MIQE (minimum information for publication of quantitative real-time PCR experiments) guideline. The whole analysis process is addressed, from nucleic acids extraction to data management. Simple answers are given, taking into account the technical constraints from clinical research in order to allow a realistic application of this guideline.

General guidelines for profiling miRNAS in cell-free body fluids and microvesicles, by NGS and qRT-PCR. Sample processing, RNA extraction and miRNAs detection procedures are detailed

Gene expression analysis of microRNA molecules is becoming increasingly important. In this study we assess the use of the mean expression value of all expressed microRNAs in a given sample as a normalization factor for microRNA real-time quantitative PCR data and compare its performance to the currently adopted approach. We demonstrate that the mean expression value outperforms the current normalization strategy in terms of better reduction of technical variation and more accurate appreciation of biological changes.