Protocols used in Molecular Biology

Protocols used in Molecular Biology

Protocols used in Molecular Biology is a compilation of several examples of molecular biology protocols. Each example is presented with a concise introduction, materials and chemicals required, a ...
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Molecular Techniques for Genotyping

Pp. 74-88 (15)

Shalini Gupta, Somali Sanyal, Suresh Kumar Yadav and Madan Lal Brahma Bhatt

Abstract

Genotyping is a process of determining the genetic constituent/genetic makeup “genotype” of an organism by examining the individual DNA sequence and comparing to a reference or other individual sequence. It helps the researchers to explore the genetic constitution, genetic linkages or variations like Single Nucleotide Polymorphisms (SNP) or multi-nucleotide changes in DNA. Identification of genotypes is also useful for determining their role in phenotypic expressions. Genotyping is an essential tool for researchers to find out disease-associated genes and gene variants. Genotype determined can also be used for the identification of susceptibility and prognosis for any disease and to find out responders/non-responders for a specific treatment, thus leading the way towards personalized medicine. Several molecular techniques have provided swift, reliable and accurate ways for determining genotypes. The process of genotyping involves molecular techniques like isolation and quantification of genomic DNA, visualization of DNA on agarose/polyacrylamide gel using electrophoresis, polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), random amplified polymorphic detection (RAPD) of genomic DNA, amplified fragment length polymorphism (AFLP), sequencing, allele-specific oligonucleotide (ASO) probes, microarrays etc. The present chapter will describe the protocols for different molecular techniques that are used to determine genotypes.

Keywords:

CTAB, DNA isolation, Genomic DNA, Genotype, Oligonucleotide, Plants, Protocol, PCR, Polymorphism, Polymerase chain reaction, RAPD, RFLP, SNP.

Affiliation:

Department of Oral Pathology and Microbiology, King George’s Medical University, Lucknow- 226003, India


Association of FOXP3 polymorphisms with susceptibility to multiple sclerosis: a meta-analysis on genetic association studies

(E-pub Abstract Ahead of Print)

Author(s): Nazanin Mousavi, Seyyed Amir Yasin Ahmadi, Zahra Mahmoudi, Reza Nekouian, Bijan Ansari-moghaddam, Farhad Shahsavar*.

Journal Name: Current Pharmacogenomics and Personalized Medicine
Formerly Current Pharmacogenomics

Abstract:

Objectives: FOXP3 is a gene related to regulatory T cells existing on chromosome X. This meta-analysis on genetic association studies is conducted to investigate association of FOXP3 polymorphisms with susceptibility to multiple sclerosis (MS).

Methods: All genetic association studies covering both FOXP3 and multiple sclerosis terms were searched in PubMed, Web of Science and Google Scholar. The information of genotype frequencies were summarized and synthesis of the results was through odds ratio (OR). Heterogeneity and publication bias were investigated using I2 scale and Begg's funnel plot respectively.

Results: For rs3761546 -3279 C/A polymorphism, AA/AY genotypes was a risk factor in comparison to CC/CY genotypes (P =0.022; OR =1.752; 95% confidence interval [CI] =1.084-2.830; random). AC genotype was a risk factor in comparison to CC/CY genotypes (P =0.004; OR =1.537; 95% CI =1.145-2.062; random) and in comparison to homozygote genotypes (P =0.016; OR =1.216; 95% CI =1.038-1.426; fixed). For rs2232365 -924 G/A polymorphism, 2 significant associations were found according to fixed effect model; of course they were not remained significant in random effect model.

Conclusion: According to the collected populations, susceptibility to and protection from MS is associated with rs3761546 -3279 C/A upstream polymorphism. However it should be regarded that this association is ethnicity dependent.

Keywords: Multiple sclerosis, FOXP3, Neuroimmunology, Meta-analysis, Genetic association study, Genetic susceptibility

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(E-pub Abstract Ahead of Print)
DOI: 10.2174/1875692118666200122163559
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Protocols used in Molecular Biology

Protocols used in Molecular Biology

Protocols used in Molecular Biology is a compilation of several examples of molecular biology protocols. Each example is presented with a concise introduction, materials and chemicals required, a ...
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2D-DIGE A Powerful Tool for Proteome Analysis

Pp. 67-73 (7)

Sudhir K. Shekhar, Jai Godheja and Dinesh Raj Modi

Abstract

In the recent past, two dimensional gel electrophoresis has emerged as a powerful molecular biology tool for the comparative expression profiling of complex protein sample. It involves the separation as well as the resolution of diverse proteins sample on the basis of isoelectric points and molecular mass of protein in two dimension ways. In this way, it reflects the view of overall proteome status including differentiation in protein expression levels, post-translational modifications etc. Moreover, this allows the identification of novel biological signatures, which may give a particular identity of pathological background to cells or tissues associated with various types of cancers and neurological disorders. Therefore, by utilizing such tools, one can clearly investigate and compare the effects of particular drugs on cells of tissues and also one can analyze the effects of disease on the basis of variations in protein expression profile at broad spectrum. Recently, to get more error-less and accurate proteome profile, conventional 2-D gel electrophoresis has been enhanced with the inclusion of different types of protein labeling dyes which enables a more comparative analysis of diverse protein sample in a single 2-D gel. In this advanced technique (2-D-DIGE), protein samples are labeled with three different types of CyDyes (Cy2, Cy3, and Cy5) separately and combined and further resolved on the same gel. This will facilitate the more accurate spot matching on a single gel platform and will also minimize the experimental variations as commonly reported in the conventional 2D-gel electrophoresis. Therefore, in the present proteomic research era, 2D-DIGE has proved to be an extremely powerful tool with great sensitivity for up to 125 ng of proteins in clinical research volubility especially, neurological and cancer related disorders.

Keywords:

2-D-DIGE, Cancer, Cy Dyes, Drugs, Electrophoresis, Molecular biology, Molecular mass, Neurological disorder, Proteome, Protein expression, Tissues.

Affiliation:

Department of Biotechnology, Babasaheb Bhimrao Ambedkar University (A Central University), Vidya Vihar, Raibareilly Road, Lucknow-226025 (U.P.), India.


Delivery Efficacy Differences of Intravenous and Intraperitoneal Injection of Exosomes:Perspectives from Tracking Dye Labeled and MiRNA Encapsulated Exosomes

(E-pub Abstract Ahead of Print)

Author(s): Xueying Zhou, Zhelong Li, Wenqi Sun, Guodong Yang, Changyang Xing, Lijun Yuan*.

Journal Name: Current Drug Delivery

Abstract:

Exosomes are cell-derived nanovesicles that play vital roles in intercellular communication. Recently, exosomes are recognized as the promising drug delivery vehicles. Up to now, how the in vivo distribution of exosomes is affected by different administration routes has not been fully understood. In the present study, in vivo distribution of exosomes following intravenous and intraperitoneal injection approaches were systemically analyzed by tracking the fluorescence labeled exosomes and qPCR analysis of C. elegans specific miRNA abundance delivered by exosomes in different organs. The results showed that exosomes administered through tail vein were mostly taken up by liver, spleen and lungs while exosomes injected intraperitoneally were more dispersedly distributed. Besides the liver, spleen, and lungs, intraperitoneal injection effectively delivered exosomes into the visceral adipose tissue, making it a promising strategy for obesity therapy. Moreover, the results from fluorescence tracking and qPCR were slightly different, which could be explained by systemic errors. Together, our study reveals that different administration routes cause significant differential in vivo distribution of exosomes, suggesting that optimization of the delivery route is prerequisite to obtain rational delivery efficiency in detailed organs.

Keywords: Exosomes, in vivo delivery, administration route, efficiency, intraperitoneal injection, intravenous injection

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(E-pub Abstract Ahead of Print)
DOI: 10.2174/1567201817666200122163251
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Glutaryl melatonin niosome gel for topical oral mucositis: anti-inflammatory and anticandidiasis

(E-pub Abstract Ahead of Print)

Author(s): Teerasak Damrongrungruang*, Panjaree Panpitakkul, Jirachaya Somudorn, Pimpitchaya Saengchart, Pramote Mahakunakorn, Prangthip Uthaiwat, Jureerat Daduang, Panyada Panyatip, Pleontip Puthongking, Aroonsri Priprem.

Journal Name: Current Drug Delivery

Abstract:

Background: Glutaryl melatonin, which is synthesized from melatonin and is a pineal gland-derived neurohormone with anti-inflammatory and anti-oxidant properties, was comparatively investigated for its potential use as a topical anti-inflammatory agent.

Objective: Glutaryl melatonin, synthesized and screened for in vitro anti-candidiasis and in vitro and in vivo anti-inflammatory activities, was formulated as a niosome gel for topical oral evaluation in 5-fluorouracil-induced oral mucositis in mice.

Method: In vitro anti-fungal activity in Candida albicans, in vitro anti-inflammatory activity in Escherichia coli liposaccharide-induced RAW cells and in vivo anti-inflammatory activity using a croton oil-induced ear edema model in ICR mice were investigated. Mucositis in mice (n = 6/group, 10-week-old mice) was induced by intraperitoneal injections of 5-fluorouracil, and the mice were subjected to a topical oral application of niosome gel containing melatonin (2% w/w) or glutaryl melatonin (2% w/w) and were compared with mice subjected to blank, fluocinolone acetonide (0.5% w/w) and control conditions.

Results: Glutaryl melatonin, at a 14. 2 mM concentration, showed the highest fungicidal effect on C. albicans using the broth dilution method, indicating a nonsignificant difference for 1 M of nystatin (p = 0.05). Nitric oxide, interleukin-6 and tumor necrosis factor-α were analyzed by ELISA. Liposaccharide-induced RAW cells were significantly reduced by glutaryl melatonin (p < 0.01). Ear edema inhibition of glutaryl melatonin was significant 1 h after application compared with that of melatonin (p = 0.03). Food consumption and body weights of the 5-fluorouracil-treated mice were significantly lower than those of the normal mice before all treatments (p < 0.05). Differences in the amount of licking behavior, which was observed in the control group for 5 min, were noticeable in the 5-fluorouracil-treated mice but not in the mice treated with the glutaryl melatonin niosome gel.

Conclusion: Glutaryl melatonin exhibited mild anti-candidiasis and anti-inflammatory properties. The incorporation of glutaryl melatonin in a niosome gel formulation, demonstrated the potential for topical oral applications to reduce oral discomfort caused by 5-fluorouracil treatment in mice.

Keywords: glutaryl melatonin, anti-candidiasis, anti-inflammation, 5-fluorouracil , niosome gel, anti-fungal

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(E-pub Abstract Ahead of Print)
DOI: 10.2174/1567201817666200122162545
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Protocols used in Molecular Biology

Protocols used in Molecular Biology

Protocols used in Molecular Biology is a compilation of several examples of molecular biology protocols. Each example is presented with a concise introduction, materials and chemicals required, a ...
[view complete introduction]

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Protocols for the Detection and Proteome Analysis of the Yellow Mosaic Virus Infected Soyabean Leaves

Pp. 60-66 (7)

Bapatla Kesava Pavan Kumar and Surapathrudu Kanakala

Abstract

Soybean (Glycine max) is one of the legumes, susceptible to yellow mosaic disease caused by Mungbean yellow mosaic India virus (MYMIV) and Mungbean yellow mosaic virus (MYMV) infection. The quantitative proteomic analysis allows achieving deeper knowledge about the viral infection. For quantitative proteomic analysis, two-dimensional gel electrophoresis (2D-PAGE) is the common method of choice. Optimization is required even for the published protocols based on the type of sample to be analyzed and for the proteins of interest. We compared four different published protocols with some modifications and selected the one which is more effective in terms of resolution and reproducibility of 2D-PAGE. Here we present our simple and cost-effective procedure for the detection of viral infection and proteomic analysis of YMV infected soybean leaves without compromising the resolution and reproducibility of 2D-PAGE.

Keywords:

Glycine max, Leaf proteome, Mungbean yellow mosaic India virus, Protein extraction, Soybean, Two-dimensional gel electrophoresis, Yellow mosaic disease.

Affiliation:

Institute of Plant Sciences, Agricultural Research Organization, Volcani Center, Israel.


In silico-based approach to investigate the ability of PEGylated rapamycin to inhibit Galectin-3

(E-pub Abstract Ahead of Print)

Author(s): Marwan Abdelmahmoud Abdelkarim Maki, Palanirajan Vijayaraj Kumar*, Manogaran Elumalai, Omer Bayazeid.

Journal Name: Current Drug Discovery Technologies

Abstract:

Galectin-3 (Gal-3) is a binding protein known to play a role in cancer and fibrosis, also implicated in various diseases of lung, liver, kidney, and heart. In this study, we have investigated the ability of modified rapamycin (RP) to bind to the carbohydrate recognition domain of Gal-3. Briefly, various molecular weights of methoxy polyethylene glycols (MPEG) were conjugated with RP to obtain RP-MPEG compounds with molecular weights of 1002.29, 1090.40, 1178.51, 1266.6 and 1354.72 g/mol. Furthermore, the molecules were docked with Gal-3 using MOE.2014 software. According to the results obtained from the molecular modeling algorithm based on shape complementarity principles, RP-MPEG with the molecular weight of 1178.51 g/mol and a logP value of 3.79 has the best affinity for a non-carbohydrate-based Gal-3 inhibitor. Moreover, in vitro hemagglutination assay results revealed that RP analog, everolimus, has possessed potent agglutination inhibition with a minimum inhibitory concentration of 160.77 ± 2.52 μg/mL, which suggested that RP derivatives are potential Gal-3 inhibitors.

Keywords: PEGylation, Rapamycin, Everolimus, Galectin-3, Molecular modeling, Cancer

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DOI: 10.2174/1570163817666200122162042
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Freeze-dried clopidogrel loaded lyotropic liquid crystal: Box-behnken optimization, in-vitro, and in-vivo evaluation.

(E-pub Abstract Ahead of Print)

Author(s): Galal M ElMahrouk, Ghada Abdelbary, Eman Abdel Hakeem*, Mahmoud H Teaima.

Journal Name: Current Drug Delivery

Abstract:

Background: Clopidogrel (CLP) suffers from extensive first pass metabolism results in a negative impact on its oral systemic bioavailability. Cubosomes are lyotropic liquid crystalline (LLC) nano-systems comprising monoolein, a steric stabilizer and an aqueous system, it considered a promising carrier for different pharmaceutical compounds. Box–Behnken design (BBD) is an efficient tool for process analysis and optimization skipping forceful treatment combinations.

Objective: The study was designed to develop freeze-dried clopidogrel loaded LLC (cubosomes) for enhancement of its oral bioavailability.

Method: A 33 BBD was adopted, the studied independent factors were glyceryl monooleate (GMO lipid phase), Pluronic F127 (PL F127steric stabilizer) and polyvinyl alcohol powder (stabilizer). Particle size (PS), polydispersity index (PDI) and zeta potential (ZP) were set as independent response variables. Seventeen formulae were prepared in accordance with the bottom up approach and in-vitro evaluated regarding PS, PDI and ZP. Statistical analysis and optimization were achieved using design expert software®, then the optimum suggested formula was prepared, in-vitro revaluated, freeze-dried with 3% mannitol (cryoprotectant), solid state characterized and finally packed in hard gelatin capsule for comparative in-vitro release and in-vivo evaluation to Plavix®.

Results: Results of statistical analysis of each individual response revealed a quadratic model for PS and PDI where a linear model for ZP. The optimum suggested formula with desirability factor equal 0.990 consisting of (200 mg GMO, 78.15 mg PL F127 and 2% PVA). LC/MS/MS study confirmed significant higher Cmax, AUC0-24h and AUC0-∞ than that of Plavix®.

Conclusion: The results confirm the capability of developed carrier to overcome the low oral bioavailability.

Keywords: Clopidogrel, Cubosomes, box-behnken, Oral bioavailability, Plavix®, LC/MS/MS

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DOI: 10.2174/1567201817666200122161433
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Protocols used in Molecular Biology

Protocols used in Molecular Biology

Protocols used in Molecular Biology is a compilation of several examples of molecular biology protocols. Each example is presented with a concise introduction, materials and chemicals required, a ...
[view complete introduction]

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Immunohistochemistry as an Important Technique in Experimental and Clinical Practices

Pp. 44-59 (16)

Hareram Birla, Sachchida Nand Rai, Saumitra Sen Singh, Walia Zahra, Neeraj Tiwari, Aijaz A. Naik, Anamika Misra, Shikha Bharati and Surya Pratap Singh

Abstract

Immunohistochemistry (IHC) is a well-known technique in the field of biological and medical sciences. This technique is based on the principle of antigenantibody interaction and is used for identification of cellular or tissue constituents, i.e., an antigen by using a specific antibody. The binding of an antibody to an antigen is confirmed either by labelled primary antibody itself or by using secondary labelling method such as fluorescence labelled antibody. Such interactions give information about the cellular process occurring inside the cell. In last few years, huge amount of data have been generated using IHC. Furthermore, adequate knowledge of this technique is required for the optimum result and its reproducibility. The detailed information about the tissue section, antigen retrieval (AR), increased sensitivity of the detection systems and proper standardization are the key points for this technique. This protocol will address overview of the technique, tissue preparation, microtome, antigen retrieval, antibodies and antigen fixation, detection methods, background reduction and trouble shootings.

Keywords:

Antibody, Antigen, DAB, Diagnosis, Enzyme, Fixation, Formaldehyde, Fluorescence, GFAP, Histology, IHC, Immunofluorescence, Microtome, Pathology, Tissue.

Affiliation:

Department of Biochemistry, Institute of Science, Banaras Hindu University, Varanasi-221005, India.

Protocols used in Molecular Biology

Protocols used in Molecular Biology

Protocols used in Molecular Biology is a compilation of several examples of molecular biology protocols. Each example is presented with a concise introduction, materials and chemicals required, a ...
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A Modified Western Blot Protocol for Enhanced Sensitivity in the Detection of a Tissue Protein

Pp. 35-43 (9)

Sachchida Nand Rai, Mallikarjuna Rao Gedda, Walia Zahra, Hareram Birla, Saumitra Sen Singh, Payal Singh, Neeraj Tiwari, Rakesh K. Singh and Surya Pratap Singh

Abstract

Western blots (WB) are designed to investigate protein levels and their patterns of modification in homogenized tissue samples. Although, Western blots are quantifiable, unlike immunohistochemistry, cellular integrity is lost. The availability of antibodies against the protein and their patterns of modification of interest form the basis of both Western blots and Immunohistochemistry. Antibodies can also be directed not only against proteins but against chemical modifications of the proteins too, such as phosphorylation and glycosylation of specific amino acid residues. In Western blotting, the proteins in the sample are denatured, size-separated on a denaturing acrylamide gel, and transferred to a nylon membrane. Antibody paratopes can then bind to the antigenic epitope in the protein present on the nylon membrane. Thus, with the help of a chemiluminescent assay system that darkens X-ray films, the resulting antibody-antigen complex can be visualized. Because of the ubiquitous and relatively inexpensive availability of WB equipment, the quality of WB in publications and following analysis and investigation of the data can be variable, possibly resulting in forged conclusions. This may be because of the poor laboratory technique and/or lack of understanding of the significant steps involved in WB and what quality control procedures should be followed to ensure effective data generation. The present book chapter focuses on providing a detailed description and critique of WB procedures and technicalities, from sample collection through preparation, blotting, and detection, to examination of the data collected. We aim to provide the reader with the improved expertise to decisively carry out, assess, and troubleshoot the WB process, in order to produce reproducible and reliable blots.

Keywords:

Western Blot, Immunohistochemistry, Antibody, Protein, Membrane.

Affiliation:

Department of Biochemistry, Institute of Science, Banaras Hindu University, Varanasi, India.