485226
Descripción
Mapa Mental por Lydia Buckmaster, actualizado hace más de 1 año
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Creado por Lydia Buckmaster
hace más de 10 años
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Transcriptomics
- Northern Blot
- Analytical technique
- Studies gene
expression by
detection of RNA in
a sample
- Useful to observe cellular control over structure and
function by determining gene expression levels in
abnormal (e.g. differentiation, morphogenesis) and
diseased conditions
- Electrophoresis
- Separates RNA
samples by size
- Detection with a
hybridisation probe
- Probe is a section of nucleotides
complementary to part of (or the
entirety of) the target sequence
- Probe is a section of nucleotides
complementary to part of (or the
entirety of) the target sequence
- Detection with a
hybridisation probe
- Separates RNA
samples by size
- Time consuming
and challenging
- Analytical technique
- Microarrays
- Most common
method of
transcriptomics
- Carries out many
genetic tests at
the same time, for
thousands of
genes in parallel
- mRNA molecules
for each gene of
interest are collected
for both the 'normal'
and 'abnormal' cell
- Hybridisation probes
for the genes of
interest are placed
onto a microarray slide
- cDNA molecules
are then generated
from the mRNA
- Each sample is
labelled with
fluorescently
labelled nucleotides
- Normal and abnormal cDNA
molecules are labelled with
different colours
- Normal and abnormal cDNA
molecules are labelled with
different colours
- Labelled cDNA
molecules hybridise to
their complementary
probes on the slide,
leaving their
fluorescent tag
- A scanner can be
used to measure
fluorescence
intensity for each
spot on the slide
- A scanner can be
used to measure
fluorescence
intensity for each
spot on the slide
- If a particular gene is
very active, it produces
more mRNA, and
therefore more cDNA
- This means more
molecules hybridise to the
slide, generating a very
bright fluorescent spot
- Less active genes
produce a dimmer
fluorescent spot
- Inactive genes
produce no mRNA, so
there is fluorescence
- This means more
molecules hybridise to the
slide, generating a very
bright fluorescent spot
- Each sample is
labelled with
fluorescently
labelled nucleotides
- cDNA molecules
are then generated
from the mRNA
- Hybridisation probes
for the genes of
interest are placed
onto a microarray slide
- Example
- Study of tumour cells
- Tumour cells are
labelled with a red dye,
and normal cells are
labelled with a green dye
- When co-hybridising
samples together, they
compete to bind to the
complementary probe
- As a result, a red spot means
that the gene is more
expressed in cancer cells
(up-regulated in cancer), and a
green spot means that it is
more expressed in normal cells
(down-regulated in cancer)
and a yellow spot means that
the gene is equally expressed
in normal and cancer cells
- Duplication of
the nucleotide
sequences in
the genome is a
real problem
- 37% of genes are highly
conserved, with a further
10% being partially
conserved, meaning that a
labelled target sequence
could hybridise to different
probes
- Better solutions are
gene-specific tags, or using
a GeneChip to quickly
determine which genes are
present in a sample
- 37% of genes are highly
conserved, with a further
10% being partially
conserved, meaning that a
labelled target sequence
could hybridise to different
probes
- Study of tumour cells
- Most common
method of
transcriptomics
- Mass Transcript Profiling
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