BioTechniques最新文献

RNA-sequencing (RNA-seq) technologies have advanced exponentially in recent years, however, the application of RNA-seq to Mycobacterium tuberculosis remains limited. We present a wet-lab and computational protocol for RNA-seq based transcriptomics that was tested on 12 replicates each of 11 clinical isolates of M. tuberculosis (n = 132) grown in vitro with and without pyrazinamide exposure. This RNA extraction method uses low-volume cultures, mechanical lysis, TRIzol^™ phase separation, and column-based purification to produce high yields of pure, intact RNA followed by rRNA depletion and cDNA library preparation. The detection of unique transcripts was optimized at a sequencing depth of 15 million reads. This method detected differential RNA expression in experimental sets with and without pyrazinamide exposure, demonstrating that the method is suitable for RNA-seq applications.

In 2006, a PCR method was introduced to subtype Blastocystis by Sanger sequencing of an ≈610 bp amplicon of the 18S rRNA gene. This method, known as barcoding-PCR, has become widespread, although the primer pair used can amplify non-Blastocystis sequences, which can result in false positives. Barcoding-PCR is most effective with DNA extracted from Blastocystis cultures, limiting its sensitivity when used directly with stool samples. As a result, barcoding-PCR can sometimes yield negative results for stool samples confirmed as Blastocystis-positive by microscopy. To improve subtyping from stool-derived DNA, we developed a Semi-Nested barcode PCR that amplifies the barcoding region in a second reaction. Our study shows that this Semi-Nested approach outperforms classical barcoding-PCR, detecting Blastocystis more reliably from stool samples with stronger gel signals and no false positives. This was confirmed by near-complete concordance (68/70 samples) with the Santin-PCR coupled to Next-Generation Sequencing (NGS) as reference standard for Blastocystis subtyping. Of particular interest, one amplicon matched the only previous report of ST35, marking this as the second global detection of ST35 and the first in Colombia. Overall, Semi-Nested barcoded PCR offers a more robust and sensitive alternative compared to classical barcoding-PCR for subtyping Blastocystis directly from stool samples.

Gene expression assays that are based on quantitative real-time PCR (qRT-PCR) method are still very popular, therefore, we developed qDATA, an open-source R-based bioinformatics application that offers a quick and intuitive analysis of raw cycle threshold (Ct) values. The application relies on a straightforward data input consisting in Ct values and on other mandatory fields specifying the experimental and control groups. qDATA automatically performs descriptive statistics, normality and statistical testing on 2^-ΔCt (or ΔCt) and 2^-ΔΔCt terms calculated with Livak's method. We also propose a qRT-PCR data analysis framework that depends on performing exhaustive ΔCt calculations within discrete biological replicates (BRs) and subsequently using the Livak formula for the complete sets of available data. These prerequisites arguably lead to an improved data analysis and statistical relevance. The efficiency of our computing approach was tested using input Ct values corresponding to immune related gene expression evaluated in experimental infection of Drosophila melanogaster and Apis mellifera workers. The presented results reveal that our working strategy is reliable and highlight the efficacy and performance of qDATA application.

Current dorsal skin flap window chambers with flat glass windows are compatible with optical coherence tomography (OCT) and multiphoton microscopy (MPM) imaging. However, light sheet fluorescence microscopy (LSFM) performs best with a cylindrical or spherical sample located between its two 90° objectives and when all sample materials have the same index of refraction (n). A modified window chamber with a domed viewing window made from fluorinated ethylene propylene (FEP), with n similar to water and tissue, was designed. In vitro imaging of collagen gels and microsphere phantoms with and without the dome showed small decreases in signal strength and image resolution due to the dome. Using a custom mouse platform for stabilization and anesthesia support, in vivo multimodality imaging with OCT, MPM, and LSFI was demonstrated.

Microsatellites are present in mitochondria, chloroplast, and nuclear DNA, but nuclear microsatellites are more useful genetic tools than those in plastids or mitochondria. Plastid and mitochondrial microsatellites have been identified in the model plant Marchantia polymorpha (liverwort), but no laboratory has published information on nuclear microsatellite loci. The aim of this study was to detect novel nuclear markers in the most commonly employed liverwort species, design PCR primers that would allow amplification, and characterize the subsequently generated loci. We detected 18 polymorphic nuclear loci in M. polymorpha, amplifiable by PCR across all chromosomes. Additionally, trans-specific amplification of the eighteen loci was characterized in the closely related taxa Marchantia emarginata and Marchantia paleacea. All loci were present in M. paleacea, whereas 17 of 18 primer pairs were amplified in M. emarginata.

Tyrosinase (TYR) inhibitors are required to treat skin hyperpigmentation. Currently live cell-based TYR assays and mushroom TYR in vitro assays are the common methods used to screen for TYR inhibitors. However, these methods are either time consuming and expensive or are not human TYR (hsTYR) specific. Here, we describe a simple hsTYR assay using cell lysate prepared from pigmented human melanoma cell lines that takes less than 3 hours to complete after collecting cell pellets. We confirmed the assay is species specific by using a known hsTYR inhibitor, kojic acid, as a positive control, while arbutin, which inhibits mushroom TYR, but not hsTYR, was not effective. This assay is a simple method to confirm hsTYR inhibition before conducting follow-up studies in live biological models.