|
qPCR & RT-qPCR Applications |
|
microRNA applications in
qRT-PCR
In genetics, a miRNA (micro-RNA) is a form of
single-stranded RNA which is typically 20-25
nucleotides long, and is thought to regulate
the expression of other genes. miRNAs are RNA genes which are
transcribed from DNA, but are not
translated into protein. The DNA
sequence that codes for an miRNA gene is
longer than the miRNA. This DNA sequence
includes the miRNA sequence and an
approximate reverse complement. When this
DNA sequence is transcribed into a
single-stranded RNA molecule, the miRNA
sequence and its reverse- complement base
pair to form a double stranded RNA hairpin
loop; this forms a primary miRNA structure
(pri-miRNA). Most microRNAs in animals
are thought to function through the
inhibition of effective mRNA translation of
target genes through imperfect base-pairing
with the 3'-untranslated region (3'-UTR) of
target mRNAs. However, the underlying
mechanism is poorly understood. MicroRNA
targets are largely unknown, but estimates
range from one to hundreds of target genes
for a given microRNA, based on target
predictions using a variety of
bioinformatics. The function of miRNAs appears to
be in gene regulation. For that
purpose, a miRNA is complementary to a part
of one or more messenger RNAs (mRNAs).
Animal miRNAs are usually complementary to a
site in the 3' UTR whereas plant miRNAs are
usually complementary to coding regions of
mRNAs. The annealing
of the miRNA to the mRNA then inhibits
protein translation, but sometimes
facilitates cleavage of the mRNA. This is
thought to be the primary mode of action of
plant miRNAs. In such cases, the formation
of the double-stranded RNA through the
binding of the miRNA triggers the
degradation of the mRNA transcript through a
process similar to RNA interference (RNAi),
though in other cases it is believed that
the miRNA complex blocks the protein
translation machinery or otherwise prevents
protein translation without causing the mRNA
to be degraded.
http://microRNA.Gene-Guantification.info
|
|
High Resolution Melting (HRM)
HRM is
a novel, homogeneous, close-tube, post-PCR
method, enabling genomic researchers to
analyze genetic variations (SNPs, mutations,
methylations) in PCR amplicons. It goes
beyond the power of classical melting curve
analysis by allowing to study the thermal
denaturation of a double-stranded DNA in
much more detail and with much higher
information yield than ever before.
HRM characterizes nucleic acid samples
based on their disassociation (melting)
behavior. Samples can be discriminated
according to their sequence, length, GC
content or strand complementarity. Even
single base changes such as SNPs (single
nucleotide polymorphisms) can be readily
identified. The
most important High Resolution Melting
application is gene scanning - the search
for the presence of unknown variations in
PCR amplicons prior to or as an alternative
to sequencing. Mutations in PCR products are
detectable by High Resolution Melting
because they change the shape of DNA melting
curves. A combination of new-generation DNA dyes,
high-end instrumentation and sophisticated
analysis software allows to detect these
changes and to derive information about the
underlying sequence constellation.
HRM Applications: The
introduction of HRM has renewed interest in
the utility of DNA melting for a wide range
of uses, including:
- Mutation discovery (gene
scanning)
- SNP genotyping
- Characterization of
haplotype blocks
- DNA methylation analysis
- Species identification
- DNA mapping
|
- Screening for loss of
heterozygosity
- DNA fingerprinting
- Somatic acquired mutation
ratios
- HLA compatibility typing
- Identification of
candidate predisposition genes
- Allelic prevalence in a
population
|
http://HRM.Gene-Quantification.info
|
|
Quantitative real-time
PCR in single-cells
Single-cell molecular-biology is a
relatively new scientific branch in biology.
The first single-cell analysis were involved
in the characterization of mitochondrial DNA
in 1988. Single-cell DNA analysis, in
particular genomic DNA, is important and may
be informative in the analysis of genetics
of cell clonality, genetic anticipation and
single-cell DNA polymorphisms. Nowadays for
most scientists the quantitative
transcriptomics
in a single-cell is much more important, and
the analytical method of choice is the
quantitative real-time RT-PCR. The relative abundance of
single mRNAs and their up- or
down-regulation in a single cell, compared
to their neighbour cells, is the goal. The
need for quantitative single-cell mRNA
analysis is evident given the vast cellular
heterogeneity of all tissue cells and the
inability of conventional RNA methods, like
northern blotting, RNAse protection assay or
classical block RT-PCR, to distinguish
individual cellular contributions to mRNA
abundance differences.
All pages
presents interesting papers, sampling
technologies and links about single-cell
qRT-PCR, using micro-manipulated or
laser-capture microdissected tissue
followed by real-time RT-PCR.
http://singlecell.Gene-Quantification.info
|
|
digital PCR
(abbreviations:
digital
PCR - DigitalPCR -
dPCR - dePCR)
Digital PCR (dPCR) is a
refinement of conventional PCR methods that
can be used to directly quantify and
clonally amplify nucleic acids (including
DNA, cDNA, methylated DNA, or RNA). The key
difference between dPCR and traditional PCR
lies in the method of measuring nucleic
acids amounts, with the former being a more
precise method than PCR. PCR carries out one
reaction per single sample. dPCR also
carries out a single reaction within a
sample, however the sample is separated into
a large number of partitions and the
reaction is carried out in each partition
individually. This separation allows a more
reliable collection and sensitive
measurement of nucleic acid amounts. The
method has been demonstrated as useful for
studying variations in gene sequences - such
as copy number variants,
point mutations, and it is routinely used
for clonal amplification of samples for
"next-generation sequencing."
http://digital-PCR.Gene-Quantification.info/
|
|
small inhibiting
RNA (siRNA)
Small interfering RNA (siRNA),
sometimes known as short interfering RNA
or silencing RNA, are a class of 20-25
nucleotide-long double-stranded RNA
molecules that play a variety of roles in
biology. Most notably, it is involved in
the RNA
interference (RNAi) pathway where
the siRNA interferes
with the expression of a specific gene. In
addition to their role in the RNAi
pathway, siRNAs also act in RNAi-related
pathways, e.g. as an antiviral mechanism
or in shaping the chromatin structure of a
genome; the complexity of these pathways
is only now being elucidated. SiRNAs were
first discovered by David Baulcombe's
group in Norwich, England, as part of
post-transcriptional gene silencing (PTGS)
in plants, and published there findings in
Science in a paper titled "A species
of small antisense RNA in
posttranscriptional gene silencing in
plants." Shortly thereafter, in
2001, synthetic siRNAs were then shown to
be able to induce RNAi in mammalian cells
by Thomas Tuschl and colleagues in a
paper, "Duplexes
of 21-nucleotide RNAs mediate RNA
interference in cultured mammalian
cells." published in Nature and
Genes & Development. This discovery
led to a surge in interest in harnessing
RNAi for biomedical research and drug
development.
|
|
CNV = Copy
Number Variants = Copy Number Variation
The human genome is
comprised of 6 billion chemical bases (or
nucleotides) of DNA packaged into two sets
of 23 chromosomes, one set inherited from
each parent. The DNA encodes roughly 27,000
genes. It
was generally thought that genes were
almost always present in two copies in a
genome. However, recent discoveries
have revealed that large segments of DNA,
ranging in size from thousands to millions
of DNA bases, can vary in copy-number. Such
copy number variations (or
CNVs) can encompass genes leading to
dosage imbalances. For example, genes that
were thought to always occur in two copies
per genome have now been found to sometimes
be present in one, three, or more than three
copies. In a few rare instances the genes
are missing altogether. Differences
in the DNA sequence of our genomes
contribute to our uniqueness. These changes
influence most traits including
susceptibility to disease. It was thought
that single
nucleotide changes (called SNPs) in
DNA were the most prevalent and important
form of genetic variation. The current
studies reveal that CNVs comprise at least
three times the total nucleotide content of
SNPs. Since CNVs often encompass genes, they
may have important roles both in human
disease and drug response. Understanding the
mechanisms of CNV formation may also help us
better understand human genome evolution.
http://CNV.Gene-Quantification.info
|
|
|