Digital PCR (dPCR) has developed considerably since the publication of the Minimum Information for Publication of Digital PCR Experiments (dMIQE) guidelines in 2013, with advances in instrumentation, software, applications, and our understanding of its technological potential. Yet these developments also have associated challenges; data analysis steps, including threshold setting, can be difficult and preanalytical steps required to purify, concentrate, and modify nucleic acids can lead to measurement error. To assist independent corroboration of conclusions, comprehensive disclosure of all relevant experimental details is required. To support the community and reflect the growing use of dPCR, we present an update to dMIQE, dMIQE2020, including a simplified dMIQE table format to assist researchers in providing key experimental information and understanding of the associated experimental process. Adoption of dMIQE2020 by the scientific community will assist in standardizing experimental protocols, maximize efficient utilization of resources, and further enhance the impact of this powerful technology.

https://biblio.ugent.be/publication/8688167/file/8688172.pdf

The Digital MIQE Guidelines Update: Minimum Information for Publication of Quantitative Digital PCR Experiments for 2020.

Objectives Recent commercialization of methods for in situ hybridization using Z-pair probe/branched DNA amplification has led to increasing adoption of this technology for interrogating RNA expression in formalin-fixed, paraffin-embedded (FFPE) tissues. Current practice for FFPE block storage is to maintain them at room temperature, often for many years. Methods To examine the effects of block storage time on FFPE tissues using a number of RNA in situ probes with the Advanced Cellular Diagnostic's RNAscope assay. Results We report marked reductions in signals after 5 years and significant reductions often after 1 year. Furthermore, storing unstained slides cut from recent cases ( Conclusions We submit that the standard practice of storing FFPE tissue blocks at room temperature should be reevaluated to better preserve RNA for in situ hybridization.

Rapid Loss of RNA Detection by In Situ Hybridization in Stored Tissue Blocks and Preservation by Cold Storage of Unstained Slides

A PCR-based quantitative assay for the evaluation of mRNA integrity in rat samples.

Gene therapy is on its way to revolutionize the treatment of both inherited and acquired diseases, by transferring nucleic acids to correct a disease-causing gene in the target cells of patients. In the fight against infectious diseases, mRNA-based therapeutics have proven to be a viable strategy in the recent Covid-19 pandemic. Although a growing number of gene therapies have been approved, the success rate is limited when compared to the large number of preclinical and clinical trials that have been/are being performed. In this review, we highlight some of the hurdles which gene therapies encounter after administration into the human body, with a focus on nucleic acid degradation by nucleases that are extremely abundant in mammalian organs, biological fluids as well as in subcellular compartments. We overview the available strategies to reduce the biodegradation of gene therapeutics after administration, including chemical modifications of the nucleic acids, encapsulation into vectors and co-administration with nuclease inhibitors and discuss which strategies are applied for clinically approved nucleic acid therapeutics. In the final part, we discuss the currently available methods and techniques to qualify and quantify the integrity of nucleic acids, with their own strengths and limitations.

Nucleic acid degradation as barrier to gene delivery: a guide to understand and overcome nuclease activity.

Reliability and reproducibility of transcriptomics-based studies are dependent on RNA integrity. In microbial ecology, microfluidics-based techniques, such as the Ribosomal Integrity Number (RIN), targeting rRNA are currently the only approaches to evaluate RNA integrity. However, the relationship between rRNA and mRNA integrity is unknown. Here, we present an integrity index, the Ratio Amplicon, Ramp , adapted from human clinical studies, to directly monitor mRNA integrity from complex environmental samples. We show, in a suite of experimental degradations of RNA extracted from sediment, that while the RIN generally reflected the degradation status of RNA the Ramp mapped mRNA degradation better. Furthermore, we examined the effect of degradation on transcript community structure by amplicon sequencing of 16S rRNA, amoA and glnA transcripts. We successfully sequenced transcripts for all three targets even from highly-degraded RNA samples. While RNA degradation changed the community structure of the mRNA profiles, no changes were observed for the 16S rRNA transcript profiles. Since both RT-Q-PCR and sequencing results were obtained, even from highly degraded samples, we strongly recommend evaluating RNA integrity prior to downstream processing to ensure meaningful results. For this, both the RIN and Ramp are useful, with the Ramp better evaluating mRNA integrity in this study.

Differential ratio amplicons (Ramp ) for the evaluation of RNA integrity extracted from complex environmental samples.

Differential amplicons (ΔAmp)-a new molecular method to assess RNA integrity.

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Differential amplicons (ΔAmp)-a new molecular method to assess RNA integrity.