Download Methods to analyze RNA expression

Methods to analyze RNA expression
Is a specific RNA transcribed?
In which cells, under which condition is it
expressed?
How much is there?
Is the amount different from other cells/times?
Principle of hybridization
Which property of DNA is it based on?
1.  Tagging
2.  Denaturation
3.  Annealing/
hybridization
4.  Visualization
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Principle of hybridization
•  The probes would seek the
complementary sequence to theirs
within the RNA mixture and bind
to the RNA.
•  The probe is tagged in some ways
that allow the visualization of the
probe-RNA target hybrid.
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RNA analysis techniques
The following experimental techniques are used to measure gene
expression and are listed in roughly chronological order, starting
with the older, more established technologies.
They are divided into two groups based on their degree of
multiplexity. Multiplexity is a measure of how many different
genes can be practically assessed by a given experiment.
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RNA analysis techniques
1.  Low-to-mid-plex techniques:
–  Northern blot
–  Fluorescent in situ hybridization
–  Reverse transcription PCR (RT-PCR) and qRT-PCR
2.  Higher-plex techniques:
–  DNA microarray
–  Tiling array
–  RNA-Seq
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Northern blotting
RNA molecules are separated by gel
electrophoresis based on their size.
RNA molecules are then transferred
onto a membrane filter (= solid
support)
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Northern blotting
The filter is incubated with the tagged probe and the RNAprobe hybrids are detected.
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Northern analysis
•  Northern analysis is the oldest of all the presented
techniques
•  Northern analysis will provide you with information about
the tissue and developmental stages when the gene is
expressed.
•  Northern can be quantitative with good controls and allow
for an estimation of the mRNA length.
•  BUT Northern blots are not scalable to many genes and use
dangerous chemicals.
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Extra information
Nature Scitable on Northern and SAGE
http://www.nature.com/scitable/topicpage/gene-expression-isanalyzed-by-tracking-rna-6525038
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RNA in situ hybridization
•  Cells, tissues or entire embryos (if small) are attached to glass
slides and permeabilized.
•  Labeled probe is added directly on top of slides for hybridization.
•  After washes the RNA-probe hybrids are visualized by
microscopy.
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RNA in situ hybridization
•  One particular example of how the probe is labeled and the
hybrid detected.
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RNA in situ hybridization
Localization of Vg1 mRNA
in Xenopus oocyte
MITF in mouse embryos
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Whole-mount in situ hybridization
Pax6
mRNA in
early chick
embryos
Whole mount means that the entire
embryo is kept intact and used as the
source of RNA.
Sonic hedgehog mRNA in the chick
nervous system, gut, and limb bud
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A brain-specific RNA in a cultured mammalian
neuron
RNA in situ is an
extremely powerful
technique to identify gene
expression in different
tissues, stages, conditions,
etc. , but it is
NOT quantitative and it is
unpractical to test many
genes
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The RT-PCR technique
Amplification of cDNA
products with PCR. The
combined protocol is referred
to as RT-PCR
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Making cDNA
cDNA stands for
complementary DNA. It is
prepared using an enzyme
known as reverse
transcriptase (RT).
It makes a DNA strand
copy of the RNA.
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The PCR technique
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The RT-PCR technique
0  Protocol flow for RT-PCR
0  Provides information if a gene
is transcribed at certain times
and in certain cells.
0  NOT quantitative
http://genome.cshlp.org/
content/18/2/242/
F4.expansion
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Limitations of standard PCR
In theory, the amount of
DNA produced at every
cycle should double.
Product (T) = (Template0) x 2n
(n = # of cycles)
Theoretical
Log Target DNA
Amplification is exponential, but the
exponential increase is limited,
o 
A linear increase follows
exponential phase
o 
Eventually plateaus
Real Life
Cycle #
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What is Real-Time RT-qPCR?
•  Similar protocol as RT-PCR but rather than checking the
presence of products at the end of the reaction by gel
electrophoresis, the presence and amplification are tested at the
end of every PCR cycles (hence “real-time”).
•  Fluorescence-based detection of amplification products through
the use of a DNA-binding dye or hybridization probe.
•  Real-time RT-qPCR is used to quantify RNA by measuring the
number of cycles required to reach a set level of product.
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Intercalation (DNA binding) dyes
•  DNA binding dyes
are inexpensive
λ
λ
λ
compared to
Taq
hybridization probes
ID
ID
ID
•  EtBr is 25 times
ID
more fluorescent
ID
Taq
when bound to dsDNA .
•  SYBR Green I is
λ
λ
125 times more fluorescent
when bound to dsDNA. The amount of fluorescence emitted is
directly proportional to the amount of double-stranded nucleic
acids.
3’
5’
5’
5’
3’
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How are quantitative data collected?
Log Target DNA
Theoretical
Real Life
Detector
Cycle #
Threshold Cycle, CT (red arrow): the point at which the fluorescence
amount crosses a preset threshold level.
CT correlates strongly with the starting copy number of any given
mRNA.
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Mathematical Implications
In ideal PCR ProductT= (Template0)*2n where n=number of
cycles
•  a CT Difference of 1 = 2 fold difference in starting template
amount
•  a CT Difference of
3.3 = 10 fold difference
in starting template
amount
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How to look at the data
Estimate the CT value
for the green pair.
Which color pair
represents the sample
where the tested RNA
was the most abundant
in the initial RNA
sample? Blue or red
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qRT-PCR summary
•  qRT-PCR provides information if a gene
is transcribed at certain times and in certain cells.
•  If done well with the correct controls, it is the best quantitative
method available today.
•  But it is technically difficult and expensive if many genes need
to be tested.
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RNA analysis techniques
1.  Low-to-mid-plex techniques:
–  Northern blot
–  Fluorescent in situ hybridization
–  Reverse transcription PCR (RT-PCR)
2.  Higher-plex techniques:
–  Expressed Sequence tag (EST)
–  DNA microarray
–  RNA-Seq
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Expressed Sequence Tag (EST) protocol
•  Contrary to the two previous RTPCR protocols, the EST analysis
looks at ALL the genes
expressed in a particular tissue/
stages, etc.
•  Provides partial sequence for the
transcripts.
•  Can help identify splice variants.
Great asset in parallel with
genome sequence analysis.
•  Often refer to as
“transcriptome”
•  NOT quantitative.
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EST protocol flow
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EST analysis
Database in the GenBank sequence depository at NCBI
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Microarray technique
Microarrays can be used to identify differences in gene expression
between two samples. It is a comparative measure.You would say
that transcripts from gene X are more abundant in cell type 1
versus cell type 2.
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View of a microarray
Magnified view of a microarray
chip. Each white dot represents
the position for a specific
sequence target.
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Microarray steps by steps
The sequence of the DNA at each
spot is known, as well as where the
sequence comes from in the
genome or which gene the
sequence represents.
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Microarray steps by steps
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Microarray steps by steps
The cDNA from one
sample is labelled with a
green fluorescent dye. The
cDNA for the other
sample is labelled red. The
two labelled cDNA
samples are mixed and
hybridized together to the
same microarray.
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Microarray steps by steps
If a gene is more
transcribed in tumor
cells, then there will
be more red labelled
cDNA than green labelled
cDNA hybridized to that one gene spot on the microarray
and the spot will look mostly red.
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Microarray steps by steps
When the 2 cDNA
attach equally to the
same DNA spot the 2
colors combined to
yellow.
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Microarray technique
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Microarrays for developmental stages
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Microarray analysis of genes activated
by Xnr in the early Xenopus embryo
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Microarray analysis of genes activated
by Xnr in the early Xenopus embryo
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Microarrays and cancer
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Heatmap
Diagram output for the
microarray analysis. All
the genes with increased
expression are
regrouped, as well
as all the ones with
similar or decreased
expression.
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Microarrays
•  Very powerful and provide good relative quantitation
•  Somewhat difficult technically
•  Require well studied genomes such that microchips can be
manufactured
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RNA sequencing analysis
•  The newest technology for RNA expression analysis
•  Provides data for all the genes expressed in a particular
sample (tissues, conditions, stages, etc.)
•  Coupled with high throughput sequencing
•  Quantitative
•  Highly technical and expensive
•  Rely heavily on computational statistical analysis
(Next PowerPoint).
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