Wildlife pathogen detection: evaluation of alternative DNA extraction protocols

Accurate detection of wildlife pathogens is critical in wildlife disease research. False negatives or positives can have catastrophic consequences for conservation and disease-mitigation decisions. Quantitative polymerase chain reaction is commonly used for molecular detection of wildlife pathogens. The reliability of this method depends on the effective extraction of the pathogen’s DNA from host samples. A wildlife disease that has been in the centre of conservationist’s attention is the amphibian disease Chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd). Here, we compare the efficiency of a spin column extraction kit (QIAGEN), commonly used in Bd DNA extraction, to an alternative spin column kit (BIOKÈ) used in extractions from other types of samples, which is considerably cheaper but not typically used for Bd DNA extraction. Additionally, we explore the effect of an enzymatic pre-treatment on detection efficiency. Both methods showed similar efficiency when extracting Bd DNA from zoospores from laboratory-created cell-cultures, as well as higher efficiency when combined with the enzymatic pre-treatment. Our results indicate that selecting the optimal method for DNA extraction is essential to ensure minimal false negatives and reduce project costs.

A genome-wide circular RNA transcriptome in Rat

Circular RNAs are a novel class of non-coding RNAs that backsplice from 5' donor site and 3' acceptor sites to form a circular structure. A number of circRNAs have been discovered in model organisms including human, mouse, Drosophila, among other organisms. There are a few candidate-based studies on circular RNAs in rat, a well-studied model organism as well. A number of pipelines have been published to identify the back splice junctions for the discovery of circRNAs but studies comparing these tools have suggested that a combination of tools would be a better approach to identify high-confidence circular RNAs. The availability of a recent dataset of transcriptomes encompassing 11 tissues, 4 developmental stages and 2 genders motivated us to explore the landscape of circular RNAs in the organism in this context. In order to understand the difference among different pipelines, we employed 5 different combinations of tools to identify circular RNAs from the dataset. We compared the results of the different combination of tools/pipelines with respect to alignment, total number of circRNAs identified and read-coverage. In addition, we identified tissue-specific, development-stage specific and gender-specific circular RNAs and further independently validated 16 circRNA junctions out of 24 selected candidates in 5 tissue samples and estimated the quantitative expression of 5 circRNA candidates using real-time PCR and our analysis suggests 3 candidates as tissue-enriched. This study is one of the most comprehensive studies that provides a map of circular RNA transcriptome as well as to understand the difference among different computational pipelines in Rat.

Quantifying the invasion and migration ability of cancer cells with a 3D matrigel drop invasion assay

Metastasis is the main cause of cancer associated morbidity which will account for approximately 600,000 deaths in the USA in 2021. Defining new mechanisms that drive cancer metastasis is vital for developing new therapeutic strategies and improving clinical outcomes for cancer patients. Herein, we describe a recently established three-dimensional Matrigel drop invasion assay to measure cancer cell invasion and migration capability in vitro. This assay is a versatile and simple tool to test the ability of cells to invade and migrate, test the functional role of genes of interest in cell invasion and migration, analyze the localization of the target proteins at the cell invasion edge in situ, and screen drug effects on cancer cell invasion and migration.

Sequence-specific inhibition of reverse transcription by recombinant CRISPR/dCas13a ribonucleoprotein complexes in vitro

The clustered regularly interspaced short palindromic repeats (CRISPR) system is widely used for genome editing because of its ability to cleave specific DNA sequences. Recently, RNA-specific CRISPR systems have been reported. CRISPR systems, consisting of a guide RNA (gRNA) and a nuclease-dead form of Cas13a (dCas13a), can be used for RNA editing and visualization of target RNA. In this study, we examined whether a recombinant CRISPR/dCas13a ribonucleoprotein (RNP) complex could be used to inhibit reverse transcription (RT) in a sequence-specific manner in vitro. Recombinant Leptotrichia wadei dCas13a was expressed using the silkworm-baculovirus expression system and affinity-purified. We found that the CRISPR/dCas13a RNP complex, combined with a chemically-synthesized gRNA sequence, could specifically inhibit RT of EGFR and NEAT1, but not non-specific RNA. Thus, the CRISPR/dCas13a RNP complex can inhibit RT reactions in a sequence-specific manner. RT inhibition by the CRISPR/dCas13a system may be useful to assess target binding activity, to discriminate RNA species retaining target sequences of gRNA, or to suppress RT from undesirable RNA species.

Fluorescent-dependent Comparative Ct Method for qPCR Gene Expression Analysis in IVF Clinical Pre-Implantation Embryonic Testing

The use of quantitative PCR (qPCR) and other PCR-based methods in the field of human in-vitro fertilization (IVF) blastocoel fluid analysis can potentially be utilized for assisting clinicians in embryo selection based on specific gene expression patterns. Since typical Comparative Ct analysis utilizes one threshold for runs per gene target and requires an inherent control group, this method is inadequate for analysis of small stochastic systems, such as embryonic-derived fluid. We mathematically demonstrate analytical modifications upon the Comparative Ct qPCR workflow to incorporate a variable fluorescence threshold (utilizing only the parameters defined in the Comparative Ct method), and subsequently demonstrate the typical workflow in which this modified method can successfully quantifiably analyze embryonic blastocoel fluid qPCR analysis.