High-throughput Isolation Method Research Articles

A High Throughput Isolation Method for Phosphate-Accumulating Organisms

Hyperphosphatemia is a secondary issue associated with chronic kidney disorder. Use of phosphate binders and dialysis are the treatments for hyperphosphatemia, albeit with harmful side effects and high cost, respectively. A safer and healthier approach is attempted to administer phosphate-accumulating organisms (PAOs) from probiotics to prevent hyperphosphatemia. However, screening and isolation of PAOs are limited by inefficient enrichment of relevant metabolism and contamination. Therefore, we devised a novel strategy to isolate elite PAOs from Lactobacillus casei JCM 1134 and Bifidobacterium adolescentis JCM 1275 (previously reported PAOs). PAOs were first enriched for phosphate uptake and incubated in low-pH phosphate-free media to dormant non-PAOs, and then purified using Percoll density gradient centrifugation. Subsequently, elite PAOs were isolated from centrifuged pellet on a toluidine blue O-supplemented agar-based media. Using this technique, elite PAOs could not only be isolated, but also semi-quantitatively scored for their phosphate accumulation capabilities. Additionally, these scores correlated well with their accumulated phosphate values. The elite PAOs isolated from L. casei and B. adolescentis showed 0.81 and 0.70 [mg-phosphate/mg-dry cell], respectively (23- and 4.34-fold increase, respectively). Thus, our method can be used to successfully isolate elite PAOs, which might be of use to prevent hyperphosphatemia at early stages.

Fast and Simplified Method for High Through-put Isolation of miRNA from Highly Purified High Density Lipoprotein.

Small non-coding RNAs (miRNAs) have been implicated in a variety of human diseases including metabolic syndromes. They may be utilized as biomarkers for diagnosis and prognosis or may serve as targets for drug development, respectively. Recently it has been shown that miRNAs are carried in lipoproteins, particularly high density lipoproteins (HDL) and are delivered to recipient cells for uptake. This raises the possibility that miRNAs play a critical and pivotal role in cellular and organ function via regulation of gene expression as well as messenger for cell-cell communications and crosstalk between organs. Current methods for miRNA isolation from purified HDL are impractical when utilizing small samples on a large scale. This is largely due to the time consuming and laborious methods used for lipoprotein isolation. We have developed a simplified approach to rapidly isolate purified HDL suitable for miRNA analysis from plasma samples. This method should facilitate investigations into the role of miRNAs in health and disease and in particular provide new insights into the variety of biological functions, outside of the reverse cholesterol transport, that have been ascribed to HDL. Also, the miRNA species which are present in HDL can provide valuable information of clinical biomarkers for diagnosis of various diseases.

Fast and Simplified Method for High Through-put Isolation of miRNA from Highly Purified High Density Lipoprotein

Fast and Simplified Method for High Through-put Isolation of miRNA from Highly Purified High Density Lipoprotein

Glycomic analysis of glycans released from glycoproteins using chemical immobilization and mass spectrometry.

Protein glycosylation is one of most common protein modifications and is involved in many biological activities. N-linked and O-linked glycosylation not only represent abundant glycan modifications, but also are structurally diverse. Mass spectrometry has emerged as a major method for glycomic analysis. However, glycan extraction from proteins and glycan modification are two critical steps in glycomic analysis of glycans using mass spectrometry. In this protocol, we describe a novel and high-throughput method for isolation and modification of glycans from glycoproteins using a chemoenzymatic approach on solid-phase. Proteins are first immobilized to a solid support and unconjugated molecules are washed away; glycans, while still linked to glycoproteins on the solid support, can be treated enzymatically or chemically on solid phase for glycan derivatization. Glycans are then released from the solid support for analysis by mass spectrometry. The procedures outlined are robust and useful for high-throughput glycomic analysis from complex biological or clinical samples.

Comparison of High-throughput and Manual DNA Extraction Methods for the Qualitative and Quantitative Analysis of MON810 Maize

PCR-based methods are widely used in the European Union and in other countries for the detection, identification, and quantification of genetically modified organisms (GMOs). The preparation of good-quality DNA from plant samples for GMO detection can be a challenging task, particularly if the DNA will be used for quantitative analysis. Two DNA extraction methods, namely manual (NucleoSpin Food kit from Machery-Nagel) and high-throughput partially automated (NucleoMag Plant kit from Machery-Nagel) methods, which utilize different DNA separation principles, were used for the isolation of DNA from maize flour samples. Despite the higher DNA recovery obtained using the high-throughput isolation method, a lower PCR efficiency was achieved, most likely due to the presence of PCR inhibitors in the extracts. We found both DNA extraction methods suitable for GMO analysis.