Tuesday, 29 December 2015

Studying Gene Expression

Studying Gene Expression:

Knowing the transcriptional activity of a gene can give valuable insight to the function of the protein it encodes and to the role it plays in an organism. Gene activity in the same individual can vary from tissue to tissue, between different developmental stages, or even from morning to night time. Gene activity is influenced by the activity of other genes and the proteins they encode. Gene expression can change in response to outside factors, such as the environment or exposure of the organism to chemical substances, competitors, or pathogens.

The classical approach to measuring the activity of a gene has been to isolate messenger RNA (mRNA), design nucleic acid molecules complementary to the gene of interest, and use those to estimate the amount of mRNA of the gene of interest present at a given time in the organism. Traditionally, this has been done for one gene at a time.

Using extremely small capillaries to apply short pieces of DNA, each uniquely representing one gene. Up to 25,000 genes can be represented on a single conventional 1.5 cm x 5 cm slide. Using these microscopic arrays of DNA spots, researchers can assess the relative amount of mRNA in a sample of all 25,000 represented genes (called the target spots) in the same time that it used to take to analyse the activity of a single gene.

Such technological advances have revolutionized the way molecular bioscience is done and have sped up the rate of new discoveries. However, they have also led to the rapid acquisition of huge amounts of data that require the use of biostatistics for analysis and validation of the collected data. In practice, gene activity is assessed, by labelling mRNA that was extracted from an organism, with fluorescent dyes. The labelled mRNA, known as the “probe” is applied to the glass slide and allowed to bind to its complementary spot on the array. This process is called hybridization. Subsequently, the unbound mRNA is washed off the slide. The slide is scanned and the amount of fluorescently labelled mRNA bound to each spot is proportional to the activity of the gene it represents.

In most cases, software analysis is then used to determine how much of a signal is due to biologically relevant processes and how much is due to technical “noise”.

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Monday, 2 November 2015

Introduction to Microarray Technology

Introduction to Microarray Technology

Molecular Biology research evolves through the development of the technologies used for carrying them out. It is not possible to research on a large number of genes using traditional methods. Micro array is one such technology which enables the researchers to investigate and address issues which were once thought to be non-traceable. One can analyse the expression of many genes in a single reaction quickly and in an efficient manner. Micro-array technology has empowered the scientific community to understand the fundamental aspects underlining the growth and development of life as well as to explore the genetic causes of anomalies occurring in the functioning of the human body.

The study of gene expression profiling of cells and tissue has become a major tool for discovery in medicine. Microarray experiments allow description of genome-wide expression changes in health and disease. The results of such experiments are expected to change the methods employed in the diagnosis and prognosis of diseases. The design, analysis, and interpretation of microarray experiments require specialized knowledge that is not part of the standard curriculum of our discipline.

Whole genome sequencing projects of many species, including humans, have provided information that allows researchers to distinguish every gene in the organism. The development of microarray technology has made it possible to survey the gene expression activity of thousands of genes at the same time by using short pieces of DNA, each uniquely representing one gene, and spotting them to a solid support, such as a microscope glass slide.

“Microarray Technology” describes a set of screening tools used to study the research fields which fall under the broad term “Genomics”. These fields of research examine, in almost their entirety, a form of the genetic material or its derivatives of an organism.

History of Microarray:

The first published article to specifically use “microarrays” was Schena et al (1989) but the way in which a DNA microarray works has stemmed from the principles developed in Southern blotting techniques (Southern, 1975). These techniques use labelled nucleic acid molecules to interrogate nucleic acids attached to a solid medium via adenine-thymine and guanine-cytosine base hybridisation (Watson and Crick, 1953). For the past few years, the primary application of microarrays has been in the identification of sets of genes that respond in an extreme manner to some treatment, or that differentiate two or more tissues.

At Stanford, Dr Mark Schena initiated a new field of science - microarray technology as the first author on the Stanford team publication in the journal Science that proving that complementary DNA molecules can be immobilized on glass and used to measure gene expression in Arabidopsis thaliana.

Schena is considered the foremost authority on microarray technology. Schena was proclaimed the "Father of Microarrays" in an article written by Lloyd Dunlap, contributing editor of Drug Discovery News, in an account of Schena's pioneering work to decipher Parkinson's disease.

The methodology of microarrays was first introduced and illustrated in antibody microarrays, also referred to as antibody matrix by Tse Wen Chang in 1983 in a scientific publication. The "gene chip" industry started to grow significantly after the 1995 Science Paper by the Ron Davis and Pat Brown labs at Stanford University. With the establishment of companies, such as Affymetrix, Agilent, Applied Microarrays, Arrayit, Illumina, and others, the technology of DNA microarrays has become the most sophisticated and the most widely used, while the use of protein and peptide microarrays are expanding.

Microarrays have quickly been established as an essential tool for gene expression profiling in relation to physiology and development. When used in conjunction with classical genetic approaches and the emerging power of bioinformatics.

Definition:

Microarray is a set of DNA sequences representing the entire set of genes of an organism, arranged in a grid pattern for use in genetic testing. It is a developing technology used to study the expression of many genes at once by placing thousands of gene sequences in known locations on a glass slide called a gene chip.

It is a 2D array on a solid substrate that is usually a glass slide or silicon thin-film cell that assays large amounts of biological material using high-throughput screening miniaturized, multiplexed and parallel processing and detection methods and hence sometimes termed as a multiplex lab-on-a-chip.

Principle behind Microarray:

The principle behind microarrays is hybridization between two DNA strands, the property of complementary nucleic acid sequences to specifically pair with each other by forming hydrogen bonds between complementary nucleotide base pairs. A high number of complementary base pairs in a nucleotide sequence means tighter non-covalent bonding between the two strands. After washing off non-specific bonding sequences, only strongly paired strands will remain hybridized. Fluorescently labeled target sequences that bind to a probe sequence generate a signal that depends on the hybridization conditions (such as temperature), and washing after hybridization. Total strength of the signal, from a spot (feature), depends upon the amount of target sample binding to the probes present on that spot. Microarrays use relative quantitation in which the intensity of a feature is compared to the intensity of the same feature under a different condition, and the identity of the feature is known by its position.

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Monday, 10 August 2015

What is Russian Doll Disease ? It's Leishmaniasis !

Russian doll disease is a virus inside a parasite inside a fly

Red blood cell, complete with Leishmania parasite (Image: Eye of Science/SPL)

It’s a Russian doll of a tropical disease. Leishmaniasis, a disease that infects 12 million people worldwide, is passed to humans by sandflies infected with the Leishmania parasite.
Now it seems that in some species of the parasite, a virus hiding inside is silently helping it subvert treatment.
Leishmaniasis is a common problem in Latin America, South Asia and parts of Africa. Depending on the form the disease takes and the species of parasite, it either attacks the skin, mucous linings of the nose and mouth, or the internal organs. It’s not easy to treat.

“Treatment failure is a major challenge for doctors and researchers, says Jean-Claude Dujardin from the Institute of Tropical Medicine in Antwerp, Belgium.
Depending on the drug and the region, treatment failure rates vary, says Dujardin. In Latin America, for example, two out of five people relapse after treatment, but this can rise to 70 per cent in parts of South Asia where another species of Leishmania circulates. The most obvious explanation is that the parasite has become resistant or that people aren’t taking the drugs properly.

Infected parasite

But in Latin America at least, it looks like there’s an alternative explanation. A virus that infects the parasite is known to make the disease more severe in mice. It now seems the same applies in people.

“The parasite is already infected by the virus and it is this package that gets transferred to the sandfly,” says Dujardin, part of an international collaboration that hunted down the virus in people infected with the L. braziliensis parasite in the Amazon basin of Bolivia and Peru. Of the people whose parasites were infected with the virus, 53 per cent of them had relapsed after drug treatment. Only 24 per cent of the people whose parasites were virus-free did so.
Similar results were seen in people infected with L. guyanensis, another parasite species common in the area. There was no link between treatment success and the parasite’s resistance to the drugs the patient was given.
“You need to imagine the system like a Russian doll,” says Dujardin. The parasite multiplies within the human host cell, and then the virus lurking within it wakes up and begins interacting with the host cell, he says.
“Leishmania alone, without the virus, is already known to subvert the immune response; it seems that the virus adds another layer of subversion, leading to treatment failure,” says Dujardin.

In good company

In some ways it’s not surprising that a virus can infect a parasite. It’s often said that parasitism is the most common way of life – with more than half of all animal species on the planet living off another in some way.
But Kevin Lafferty, an ecologist at the University of California, Santa Barbara, says that although viruses are known to infect bacteria and parasites, instances of a virus infecting a parasite that in turn infects another host are not very common. “This is a fascinating piece of detective work with important implications for human health.”
However, Jorge Alvar at the Drugs for Neglected Diseases Initiative in Switzerland, cautions that we still don’t how the virus affects the evolution of the parasite, or how it ultimately impacts the patient.
But, in theory, the virus gives us an added drug target, he says. “In this case a patient could be treated with either anti-Leishmania drugs or anti-virals, or both.
Similar viruses have been found in other parasites, for example, in the diarrhoea-causing Giardia and Cryptosporidium, and in Trichomonas vaginalis that causes a sexually transmitted infection. Surveys of their prevalence could help us better understand the effect of viral infection of parasites and could play a role in how we treat these parasitic diseases, says Dujardin.

Journal reference: Journal of Infectious Diseases, DOI: 10.1093/infdis/jiv355 (L. braziliensis); DOI: 10.1093/infdis/jiv354 (L. guyanensis)

If you want to read the complete article then you may visit the (actual source) direct link ---> https://www.newscientist.com/article/dn28020-russian-doll-disease-is-a-virus-inside-a-parasite-inside-a-fly/

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How if We don’t need bodies?

What if … We don’t need bodies?

Uploading our minds onto computers could be the future. But cutting ties with our animal roots would raise ethical questions for which we don't yet have answers

Is anybody in there? (Image: Skizzomat)

MINDS result from bodies, but that link can be compromised. If I severed my spinal cord at the neck, I’d get no inputs from most of my body, says Michael Graziano, a neuroscientist at Princeton University. “But I’m still a person, I still have experience, I can still think.”
What if we could separate mind from body entirely? Many now believe that we will transfer our minds on to computers, whether in a matter of decades or hundreds of years. “I would say that it’s not only possible, it’s inevitable,” says Graziano.
What would life as an upload be like? We’d still need outside stimulation. Cut off entirely, a brain would suffer sensory deprivation, says Anders Sandberg at the University of Oxford. “It’s going to fall asleep, then hallucinate and probably gently go mad.

If you want to read the complete article then you may visit the (actual source) direct link ---> https://www.newscientist.com/article/mg22730330-600-what-if-we-dont-need-bodies/

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Animal Biotechnology - Tissue Culture and Cell Culture

Use of Antibiotics in Animal Tissue Culture for maintaining sterility

Prevention of Cross-contamination in Animal cell cultures

Maintenanceof Sterility in Animal Tissue Culture

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