Location via proxy:   [ UP ]  
[Report a bug]   [Manage cookies]                
Scribd
Upload
128 views

Transcriptomics: Shivangi Asthana B.Sc. Biotech

Transcriptomics is the study of an organism's transcriptome, which is the sum of all its RNA transcripts. Techniques used in transcriptomics include serial analysis of gene expression (SAGE), quantitative PCR (qPCR), DNA microarrays, and RNA sequencing. These techniques allow researchers to study gene expression across many genes simultaneously and provide a snapshot of the transcripts present in a cell at a given time. Protein structure and analysis techniques are also important areas of study that provide insights into gene expression and function.

Uploaded by

sachin kumar
Copyright
© © All Rights Reserved
Available Formats
Download as PPTX, PDF, TXT or read online on Scribd
128 views

Transcriptomics: Shivangi Asthana B.Sc. Biotech

Transcriptomics is the study of an organism's transcriptome, which is the sum of all its RNA transcripts. Techniques used in transcriptomics include serial analysis of gene expression (SAGE), quantitative PCR (qPCR), DNA microarrays, and RNA sequencing. These techniques allow researchers to study gene expression across many genes simultaneously and provide a snapshot of the transcripts present in a cell at a given time. Protein structure and analysis techniques are also important areas of study that provide insights into gene expression and function.

Uploaded by

sachin kumar
Copyright
© © All Rights Reserved
Available Formats
Download as PPTX, PDF, TXT or read online on Scribd
You are on page 1/ 22

TRANSCRIPTOMICS

Shivangi Asthana
B.Sc. Biotech
TRANSCRIPTOMICS
• Transcriptomics technologies are the techniques used to study an
organism’s transcriptome, the sum of all of its RNA transcripts
• The information content of an organism is recorded in the DNA o f
its genome and expressed through transcription.
• mRNA serves as a transient intermediary molecule in the information
network, whilst non-coding RNAs perform additional diverse
functions
• . A transcriptome captures a snapshot in time of the total transcripts
present in a cell
• The first attempts to study the whole transcriptome began in the early
1990s, and technological advances since the late 1990s have made
transcriptomics a widespread discipline.
• Transcriptomics has been defined by repeated technological
innovations that transform the field
SAGE
• Serial analysis of gene expression (SAGE) is a transcriptomic
technique used by molecular biologists to produce a snapshot
of the messenger RNA population in a sample of interest in
the form of small tags that correspond to fragments of those
transcripts.
Q- PCR
• Quantitative PCR (qPCR) or real-time PCR is a sensitive,
reproducible and accurate method for determining the DNA or
most commonly mRNA levels in tissues or cells.
• The amount of PCR product produced in every cycle step of
the PCR reaction is monitored, which renders possible by
fluorescent probes (e.g. TaqMan) or dyes (e.g. SYBR Green).
• In a conventional PCR reaction, the PCR product amount is
measured as an end-point analysis on an agarose gel.
• In a real-time PCR reaction, the PCR product amount can be
measured at any time of the PCR process, with the highest
precision during the exponential phase.
• The Ct-value is used in the calculation of mRNA transcript
levels
MICROARRAY
• A DNA microarray (also commonly known as DNA chip
or biochip) is a collection of microscopic DNA spots attached
to a solid surface.
• Scientists use DNA microarrays to measure
the expression levels of large numbers of genes
simultaneously or to genotype multiple regions of a genome.
• . These can be a short section of a gene or other DNA element
that are used to hybridize a cDNA or cRNA (also called anti-
sense RNA) sample (called target) under high-stringency
conditions.
• Probe-target hybridization is usually detected and quantified
by detection of fluorophore-, silver-, or chemiluminescence-
labeled targets to determine relative abundance of nucleic
acid sequences
PROTIEN ARCHITECTURE
• Protein structure is the three-dimensional arrangement of
atoms in an amino acid-chain molecule.
• Proteins are polymers— specifically polypeptides — formed
from sequences of amino acids, the monomers of the
polymer.
• A single amino acid monomer may also be called a residue
indicating a repeating unit of a polymer. Proteins form by
amino acids undergoing condensation reactions, in which the
amino acids lose one water molecule per reaction in order to
attach to one another with a peptide bond
PRIMARY STRUCTURE
• The primary structure of a protein refers to the sequence
of amino acids in the polypeptide chain.
• The primary structure is held together by peptide bonds that
are made during the process of protein biosynthesis The two
ends of the polypeptide chain are referred to as the carboxyl
terminus (C-terminus) and the amino terminus (N-terminus)
based on the nature of the free group on each extremity.
• Counting of residues always starts at the N-terminal end (NH2-
group), which is the end where the amino group is not
involved in a peptide bond.
• The primary structure of a protein is determined by
the gene corresponding to the protein
SECONDARY STRUCTURE

• Secondary structure refers to highly regular local sub-


structures on the actual polypeptide backbone chain.
• Two main types of secondary structure, the α-helix and the β-
strand or β-sheets , were suggested in 1951 by Linus Pauling
and coworkers.
• These secondary structures are defined by patterns
of hydrogen bonds between the main-chain peptide groups.
TERTIARY STRUCTURE
• The tertiary structure of a protein or any
other macromolecule is its three-dimensional structure, as
defined by the atomic coordinates.
• Proteins and nucleic acids fold into complex three-
dimensional structures which result in the molecules'
functions.
• While such structures are diverse and complex, they are often
composed of recurring, recognizable tertiary structure motifs
and domains that serve as molecular building blocks.
• Tertiary structure is considered to be largely determined by
the biomolecule's primary structure
QUATERNARY STRUCTURE
• The quaternary structure refers to the number and
arrangement of multiple protein molecules in a multi-subunit
complex.
• For nucleic acids, the term is less common, but can refer to
the higher-level organization of DNA in chromatin, including
its interactions with histones, or to the interactions between
separate RNA units in the ribosome
PROTEOMICS ANALYSIS – 2-
DE
• Two-dimensional gel electrophoresis (2-DE) is a key tool for
comparative proteomics research. In 2-DE, mixtures of
proteins are separated by charge (isoelectric point, pI) in the
first dimension and further separated by mass in the second
dimension on 2-D gels.
• Coupling 2-DE with immobilized pH gradients, IPG-Dalt, has
provided higher resolution, improved reproducibility, and
higher loading capacity for preparative purposes (O’Farrell,
1975).
• The 2-DE can achieve the separation of several thousand
different proteins in one gel.
• Stains such as Co-omassie Brilliant Blue, silver, SYPRO Ruby,
and Deep Purple can be employed to visualize the proteins
(Nilsson et al., 2000).
• Unfortunately, 2-DE technique is a time-consuming and labor-
intensive process.
• Conventional 2-DE is restricted to the detection of denatured
proteins in the size range of 10~200 kDa at pH 3.5~11.5. T
• Two-dimensional gel electrophoresis or 2D-PAGE is the
primary technique for proteomics work.
• It separates the complex mixture of samples using two
different properties of the proteins. In the first dimension,
proteins are separated by the pI value and in the second
dimension by the relative molecular weight.
DIGE
• Difference gel electrophoresis is an emerging technology that allows
for accurate quantification with statistical confidence while
controlling for non biologic variation, and also increases the dynamic
range and sensitivity of traditional 2D polyacrylamide gel
electrophoresis.
• With inclusion of an internal standard formed from equal amounts
of every sample in an experiment, difference gel electrophoresis
technology also allows for repetitive measurements and
multivariable analyses to be quantitatively analyzed in one co-
ordinated experiment, yielding statistically-significant changes in
protein expression related to many disease states.
• This technique promises to be an important tool in clinical
proteomics and the study of the mechanism of disease, investigating
diagnostic biomarkers and pinpointing novel therapeutic targets.
MASS SPECTROMETRY
• Mass spectrometry (MS) is an analytical technique that
ionizes chemical species and sorts the ions based on
their mass-to-charge ratio. In simpler terms, a mass
spectrummeasures the masses within a sample. Mass
spectrometry is used in many different fields and is applied to
pure samples as well as complex mixtures.
• A mass spectrum is a plot of the ion signal as a function of the
mass-to-charge ratio. These spectra are used to determine the
elemental or isotopic signature of a sample, the masses of
particles and of molecules, and to elucidate the chemical
structures of molecules, such as peptides and other chemical
compounds.
• In a typical MS procedure, a sample, which may be solid,
liquid, or gas, is ionized, for example by bombarding it with
electrons. This may cause some of the sample's molecules to
break into charged fragments. These ions are then separated
according to their mass-to-charge ratio, typically by
accelerating them and subjecting them to an electric or
magnetic field: ions of the same mass-to-charge ratio will
undergo the same amount of deflection
THANK YOU

You might also like