BioTechniques最新文献

Processing methods that do not induce shape distortion are essential for the analysis of tissue morphology. Although the use of agarose-gelatin double embedding to reduce shape distortion has been suggested, the effect of matrix concentration has not been addressed. Therefore, combinations of agarose (1-3%) and gelatin (1-10%) were evaluated using multicellular spheroids without extracellular matrix as a model of mechanical fragility. In this study, a blend of 2% agarose and 5% gelatin effectively prevented distortion. This protocol can enhance the morphological analysis of delicate tissues.

The genetic stability of recombinant CHO cell lines producing therapeutic proteins is critical for ensuring consistent quality in biopharma-ceutical products. Southern blotting remains the gold standard for evaluating transgene integrity and stability in these cell lines. In the biopharmaceutical industry, transposon-based expression systems are widely utilized to generate highly productive and genetically stable CHO cell lines. However, evaluating transgene integration sites and integrity in such cell lines is challenging with standard Southern blotting protocols. This difficulty arises because transposon-mediated transfection often results in multiple independent integration sites in the host genome, each typically harboring a single transgene copy. Upon restriction enzyme digestion, similar-sized DNA fragments are generated, reducing resolution and complicating the separation and detection of the transgenes using standard blotting protocols. Here, we present a modified Southern blotting protocol that significantly improves the resolution of integration banding patterns by refining key steps, including purification of digested DNA prior to electrophoresis and an enhanced DNA transfer method. This protocol was successfully applied to analyze multiple transposon-derived CHO cell lines with high transgene copy numbers, enabling more precise and efficient detection of transgene integration.

Antimicrobial resistance poses a significant challenge for infection control, requiring the development of accurate and high-throughput diagnostic techniques. We expanded and optimized an existing DNA microarray platform for the molecular characterization of vancomycin-resistant Enterococcus (VRE) by incorporating resistance, virulence, species-specific, and typing markers. The enhanced microarray allows for the simultaneous analysis of up to 96 strains, providing detailed genetic profiles of clinical isolates. VRE strains from Romania and Bavaria, Germany, were analyzed, and the results were compared to those obtained using traditional typing methods, such as multilocus sequence typing (MLST). Next-generation sequencing (NGS) was used in parallel to validate the microarray findings and explore genomic relationships. The microarray revealed considerable genetic diversity and potential epidemiological linkages among isolates. A novel hexadecimal-based nomenclature system was introduced for standardized and scalable strain classification. Comparative analysis demonstrated that the array profiles provided greater discriminatory power and practical resolution than MLST. Receiver operating characteristic (ROC) curve analysis of 187 target genes in 220 isolates gave diagnostic sensitivity and specificity of 100%. This integrated approach offers a cost-effective, rapid, and adaptable global VRE surveillance and infection control tool. It provides a practical alternative to conventional typing systems and facilitates early detection of outbreaks and emerging clones.

This study examines the stability of human mRNA and DNA in stool samples for noninvasive gastrointestinal disease detection. While stool samples are valuable for diagnosing conditions like inflammatory disorders and colorectal cancer, mRNA instability poses significant challenges, risking false-negative results. To investigate this, 97 stool samples were treated with a specialized stabilizing solution and stored at room temperature, with analyses conducted on Day 1 and Day 15. The research aimed to improve storage protocols for enhanced reliability in mRNA diagnostics, aiding in personalized medicine and biomarker discovery. Results showed variability in total nucleic acid yields, increasing from Day 1 (mean 112 ng/µL) to Day 15 (mean 165 ng/µL), highlighting the benefits of improved homogenization and bacterial lysis. Human DNA remained stable over the 14-day period. For RNA stability, three mRNA markers were analyzed: Carcinoembryonic Antigen (CEACAM5), Prostaglandin-Endoperoxide Synthase 2 (PTGS2) and cortactin (CTTN). Both CEACAM5 (p=0.064) and PTGS2 (p=0.79) maintained stability, while CTTN showed a statistically significant but only modest reduction in expression (p < 0.0001). Overall, the stabilization buffer proved effectiveness in preserving nucleic acids and provided insights into mRNA marker stability over time.

Pre-harvest sprouting, germination of the seed on the spike, causes reduced grain quality and marketability in US wheat. Methods devised to induce and study pre-harvest sprouting vary significantly from one another in procedure, induction time, and/or measurement, and often fail to accurately reflect natural sprouting, which varies over years and locations. An artificial sprouting chamber and protocol with a shorter exposure time and relative humidity more relevant to field conditions was significantly correlated (p < 0.01) with natural pre-harvest sprouting over three years for ten wheat varieties measured by alpha amylase activity. The alpha amylase activities of ten wheat varieties tested under the developed artificial sprouting chamber and protocol were significantly correlated (p = 0.001) with those subjected to overhead irrigated field sprouting tests over three years. Four pairs of the ten varieties were genetically related but showed significantly different sprouting and alpha amylase activity. These varietal pairs may prove useful in studying the genetics of pre-harvest sprouting. While the study was performed in wheat, other susceptible crops such as rice, barley, rye, and sorghum could be tested using this method.

Differentiation of osteoclasts from their macrophage precursors can be assessed via multiple methods including microscopy, gene expression analysis, and protein immunoblotting, but these methods can be expensive or labor intensive and subject to variation in approach and interpretation. We have developed a low-cost kinetic assay for the quantification of Tartrate Resistant Acid Phosphatase (TRAP), which is secreted by osteoclasts in increasing amounts as they differentiate. This assay demonstrates reliable reproducibility and provides sensitive, quantified data that accurately represents altered levels of osteoclastogenesis due to variations in Receptor Activator of Nuclear Factor κB (RANK) signaling and the presence of known osteoclast inhibitors. The assay is cost-effective, scalable and readily adoptable by labs conducting osteoclast biology research.

Effective polymerase chain reaction (PCR) product purification is essential for downstream applications like next-generation sequencing (NGS). While standard protocols remove short primers (20-30 nucleotides [nt]), NGS workflows require efficient removal of larger primers (40-50 nt) to prevent amplification artifacts. This study compared commercial kits (magnetic beads, silica columns, enzymatic degradation) with traditional isopropanol/ethanol precipitation and simple dilution for their ability to remove large primers from a 161-bp KRAS PCR product. Efficacy was assessed by a secondary PCR designed to amplify remaining primers. Optimal yield was achieved with isopropanol precipitation with NH₄Ac (2.5-3.0 M) and overnight incubation at 4 to -20 °C. Interestingly, a simple 1:200 dilution showed comparable results. Among commercial kits, magnetic beads demonstrated superior primer removal, as evidenced by a substantially lower concentration of the secondary PCR product (0.3 ng/µL ± 0.23) compared to untreated samples (22.13 ng/µL ± 1.7). For NGS workflows, magnetic beads are the most effective method for removing large primers, while isopropanol precipitation and dilution offer viable, low-cost options, particularly for workflows involving multiple PCR steps.

RNA sequencing (RNA-Seq) is an essential assay for studying transcriptome profiling. Ribosomal RNA (rRNA) comprises more than 80-90% of total cellular RNA; efficient removal is essential for accurately capturing transcriptomes, particularly to sequence low-abundance mRNAs. Inefficient rRNA removal during library preparation can result from variations in sample quality, preparation methods, and handling. Estimating rRNA content in RNA-Seq libraries pre-sequencing is therefore challenging due to the absence of a reliable and cost-effective assessment method. This study addresses the issue by introducing a scalable real-time quantitative polymerase chain reaction (qPCR) based assay targeting human 18S rRNA to evaluate rRNA depletion efficiency of RNA-Seq libraries pre-sequencing. qPCR efficiency was optimized using serial dilutions of Universal Human Reference (UHR) control, and Ct thresholds were established using pilot data from 644 libraries. Following this optimization, analysis of 1748 human Total RNA-Seq libraries and 445 Poly A + two widely used RNA-Seq library methods, demonstrated a strong correlation between 18S rRNA qPCR results and post-sequencing rRNA rates. This assay was also used to evaluate the performance of Oligo (dT) beads from four different vendors to enrich mRNA. This 18S rRNA qPCR assay is a cost-effective, scalable approach for reliably predicting rRNA read percentage in RNA-Seq libraries pre-sequencing.

Despite increasing rates of metabolic syndrome, cancer treatment regimens often ignore the underlying metabolic dysfunction in patients. A high proportion of cancer patients have metabolic dysfunction, and caloric restriction has shown the potential to improve cancer treatment response. However, preclinical efficacy is hindered by inconsistent protocols. We demonstrated that a CR (caloric restriction) diet enhances radiotherapy outcome by promoting apoptosis and downregulating cell survival pathways in mice. However, CR animal models implementation greatly differs in previous cancer studies, hindering its clinical translation. Here, we propose an effective CR protocol that safely achieves a 30% caloric reduction in two weeks. Week one involves single housing the mice and taking individual caloric intake measurements across four days. Week two involves weaning the mice to a 30% reduction by reducing their caloric intake by 10% every other day, which allows for a safe reduction. This protocol integrates individualized intake measurement, gradual weaning, and real-time monitoring-features rarely combined in prior models-offering a reproducible and translationally relevant framework for cancer studies.