Transcriptome Analysis Methods: From the Serial Analysis of Gene Expression and Microarray to Sequencing new Generation Methods

Up-to-date research in biology, biotechnology, and medicine requires fast genome and transcriptome analysis technologies to investigate cellular state, physiology, and activity. Gene expression is the process of generating messenger RNA copies of a gene. The transcriptome, which contains the mRNA of the cell, reflects the cell's overall gene expression pattern. Understanding the nature and frequency of each RNA molecule in a given cell under certain circumstances is necessary to examine the transcriptome. Microarray and serial analysis of gene expression are two primary techniques researchers use in transcriptome studies. Here, microarray technology and next-generation sequencing of transcripts are states of the art. Since microarray technology is limited to RNA, quantifying transcript levels and sequence information, RNA-Seq provides nearly unlimited possibilities in modern bioanalysis. Sequencing of RNA, or RNA-Seq, is now a standard method to analyze gene expression and uncover novel RNA species. In addition, aspects of RNA biogenesis and metabolism can be interrogated with specialized techniques for cDNA library preparation. The present study will introduce and compare new high-performance methods used in examining the transcriptome. This also presents a detailed description of next-generation sequencing, describes the impact of this technology on transcriptome analysis, and explains its possibilities to explore the modern RNA world

The Use of MALDI-TOF Mass Spectrometry Technology in Molecular Analysis of Microbial Pathogenesis

Mass spectrometry is a method of evaluation used to identify which particles compose an example based on the ions' mass spectrum. Mass spectrometers can perform conventional detection and quantitation of target analytes. However, they can also be used for the rapid discovery of bacteria within a medical setting. Matrix-assisted laser desorption/ionization-time of flight mass spectrometer is one of the most prominent MS tools applied in biology, with its robust and accurate recognition of categories and types of a wide variety of Gram-positive and negative microorganisms. Mass spectrometry detection is based on determining a particular range of each kind and matching it with a comprehensive data source within the tool. Today's study describes the history and sample preparation of the MALDI-TOF MS technique. Moreover, the applications of MALDI-TOF MS microbial recognition in the center and the presence of antimicrobial resistance will be presented. Besides, the present restrictions and future use of MALDI-TOF MS in forthcoming daily scientific practice are reviewed. In this review, microorganisms will also be addressed in future clinical applications, primarily using MALDI-TOF MS in microbiology to identify and analyze antibiotic resistance

Current Advances in DNA Methylation Analysis Methods

DNA methylation is one of the epigenetic changes, which plays a major role in regulating gene expression and, thus, many biological processes and diseases. There are several methods for determining the methylation of DNA samples. However, selecting the most appropriate method for answering biological questions appears to be a challenging task. The primary methods in DNA methylation focused on identifying the state of methylation of the examined genes and determining the total amount of 5-methyl cytosine. The study of DNA methylation at a large scale of genomic levels became possible following the use of microarray hybridization technology. The new generation of sequencing platforms now allows the preparation of genomic maps of DNA methylation at the single-open level. This review includes the majority of methods available to date, introducing the most widely used methods, the bisulfite treatment, biological identification, and chemical cutting along with their advantages and disadvantages. The techniques are then scrutinized according to their robustness, high throughput capabilities, and cost

Induction of proteome changes involved in the cloning of mcr-1 and mcr-2 genes in Escherichia coli DH5-α strain to evaluate colistin resistance

ABSTRACT: Objectives: Plasmid genes, termed mobile colistin resistance-1 (mcr-1) and mobile colistin resistance-2 (mcr-2), are associated with resistance to colistin in Escherichia coli (E. coli). These mcr genes result in a range of protein modifications contributing to colistin resistance. This study aims to discern the proteomic characteristics of E. coli–carrying mcr-1 and mcr-2 genes. Furthermore, it evaluates the expression levels of various proteins under different conditions (with and without colistin). Methods: Plasmid extraction was performed using an alkaline lysis–based plasmid extraction kit, whereas polymerase chain reaction was used to detect the presence of mcr-1 and mcr-2 plasmids. The E. coli DH5α strain served as the competent cell for accepting and transforming mcr-1 and mcr-2 plasmids. We assessed proteomic alterations in the E. coli DH5α strain both with and without colistin in the growth medium. Proteomic data were analysed using mass spectrometry. Results: The findings revealed significant protein changes in the E. coli DH5α strain following cloning of mcr-1 and mcr-2 plasmids. Of the 20 proteins in the DH5α strain, expression in 8 was suppressed following transformation. In the presence of colistin in the culture medium, 39 new proteins were expressed following transformation with mcr-1 and mcr-2 plasmids. The proteins with altered expression play various roles. Conclusion: The results of this study highlight numerous protein alterations in E. coli resulting from mcr-1 and mcr-2–mediated resistance to colistin. This understanding can shed light on the resistance mechanism. Additionally, the proteomic variations observed in the presence and absence of colistin might indicate potential adverse effects of indiscriminate antibiotic exposure on treatment efficacy and heightened pathogenicity of microorganisms

MALDI-TOF Mass Spectroscopy Applications in Clinical Microbiology

There is a range of proteomics methods to spot and analyze bacterial protein contents such as liquid chromatography-mass spectrometry (LC-MS), two-dimensional gel electrophoresis, and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS), which give comprehensive information about the microorganisms that may be helpful within the diagnosis and coverings of infections. Microorganism identification by mass spectrometry is predicted on identifying a characteristic spectrum of every species so matched with an outsized database within the instrument. MALDI-TOF MS is one of the diagnostic methods, which is a straightforward, quick, and precise technique, and is employed in microbial diagnostic laboratories these days and may replace other diagnostic methods. This method identifies various microorganisms such as bacteria, fungi, parasites, and viruses, which supply comprehensive information. One of the MALDI-TOF MS’s crucial applications is bacteriology, which helps identify bacterial species, identify toxins, and study bacterial antibiotic resistance. By knowing these cases, we will act more effectively against bacterial infections