Download single cells

RT-PCR from single-cell lysates
RT-PCR from single-cell lysates
RT-PCR from single-cell lysates
Why study single cells?
Because tissues are composed of heterogeneous mixtures of cells, gene expression measurements based on the homogenized
population don’t account for the small but critical changes occurring in individual cells.
Single-cell analysis can be critical in applications such as candidate drug screening, cell differentiation and stem cell studies, and measuring
individual cell responses to specific stimuli.
The Ambion® Single Cell-to-CT™ kit enables you to study gene expression at the single-cell level, without having to first isolate RNA. The
kit is optimized for maximum sensitivity for reliable, consistent results even when starting from a single cell. This new kit provides not only a
validated workflow for gene expression analysis but also a standardized platform for the study of single cells.
The use of a standardized platform such as the Ambion® Single Cell-to-CT™ Kit allows results from single cells to be comparable across the
research community. This will accelerate single cell–based applications such as the evaluation of biomarkers from limited clinical samples or
other precious samples.
Functional steps the Ambion® Single Cell-to-CT™ kit
1.
Cell lysis
2.
Reverse transcription
3.
cDNA pre-amplification
4.
Real time PCR
Workflow of single-cell gene profiling
Applications from cell lysates
Cell lysated
whole genome amplification (WGA)
Next Generation DNA-Sequencing
Polony Sequencing
This technique was first developed by Dr. George Church's group at Harvard Medical School.
Polony sequencing is an inexpensive but highly accurate multiplex sequencing technique that can be used to “read” millions of
immobilized DNA sequences in parallel. It combined an in vitro paired-tag library with emulsion PCR, bridge-PCR and
sequencing in fluidics system.
Unlike other sequencing techniques, Polony sequencing technology is an open platform with freely downloadable, open source
software and protocols. Also, the hardware of this technique can be easily set up with a commonly available epifluorescence
microscopy and a computer-controlled flow cell/fluidics system.
A. the paired end-tag library
construction and
template amplification
B. DNA sequencing
Polony Sequencing
Next Generation DNA Sequencing using microbreads
Emulsion PCR and Pyrosequencing
Pyrosequencing
The method allows sequencing of a single strand of DNA by synthesizing
the complementary strand along it, one base pair at a time, and detecting
which base was actually added at each step. The template DNA is
immobile, and solutions of A, C, G, and T nucleotides are sequentially
added and removed from the reaction.
The single-strand DNA (ssDNA) template is hybridized to a
sequencing primer and incubated with the enzymes DNA
polymerase, ATP sulfurylase, luciferase and apyrase, and with the
substrates adenosine 5´ phosphosulfate (APS) and luciferin. Light is
produced only when the nucleotide solution complements the first
unpaired base of the template.
1. The addition of one of the four deoxynucleoside triphosphates (dNTPs)
(dATPαS, which is not a substrate for a luciferase, is added instead of
dATP to avoid noise) initiates the second step. DNA polymerase
incorporates the correct, complementary dNTPs onto the template. This
incorporation releases pyrophosphate (PPi).
2. ATP sulfurylase converts PPi to ATP in the presence of adenosine 5´
phosphosulfate. This ATP acts as a substrate for the luciferasemediated conversion of luciferin to oxyluciferin that generates visible
light in amounts that are proportional to the amount of ATP. The light
produced in the luciferase-catalyzed reaction is detected by a camera and
analyzed in a pyrogram.
3. Unincorporated nucleotides and ATP are degraded by the apyrase, and
the reaction can restart with another nucleotide.
The templates for pyrosequencing can be made both by solid phase
template preparation (streptavidin-coated magnetic beads) and enzymatic
template preparation (apyrase+exonuclease). So Pyrosequencing can be
differentiated into two types, namely Solid Phase Pyrosequencing and
Liquid Phase Pyrosequencing.
Polony Sequencing
AAAAATCCGAC
TTTTTAGGCTG
AGGCTGTTTTT
TCCGACAAAAA
The Target DNA sequence is randomly sheared, and then the
fragments (about 1 kb in size) are selected. After making the
ends of these fragments blunt and A-tailing, in which an A is
added to the 30 ends of fragments, the fragments are
circularized, using 30 bp synthesized oligonucleotides (T30)
with two outward facing recognition sites for a type II
restriction enzyme (MmeI);
A. DNA fragmentation, A-tailing, and
T30 and two sites for MmeI addition
B. Rolling circle replication
then amplification will occur, using rolling circle replication.
In the next step, the amplified circularized DNA is subjected to
MmeI, which cuts at a distance of 17–18 bp after detecting
the recognition site, and this results in the generation of a
fragment of about 70 bp from which 30 bp belongs to T30 and
the rest belongs to two 17–18 bp flanking regions or tags.
AGGCTGTTTTT
TCCGACAAAAA
AAAAATCCGAC
TTTTTAGGCTG
~70 bp
The resulting fragments then end repaired and two emulsionPCR primers will be attached to their 30 and 50 ends, resulting
in the production of a 135 bp fragment that is then subjected to
amplification. These 135 bp fragments construct a paired insert
library.
C. Cut using MmeI and addition
of new primer for binding
magnetic beads and sequencing
~135 bp
primer-genome seq-MmeI-T30-MmeI-genome seq-primer
Rolling Cycling Amplification (RCA)
Blue lines denote target DNA sequences, green lines represent
oligonucleotide primers and red lines represent new DNA
synthesized by the polymerase.
(a) Linear template and single primer. After primer binding, the
polymerase synthesizes one complementary strand.
(b) Circular template and single primer. The polymerase
synthesizes a complementary strand beginning at the bound
primer. After one round, the primer and the synthesized strand
are displaced and DNA synthesis continues for additional
rounds. By this, a long concatemeric single-stranded DNA is
produced.
(c) Circular template and multiple random primers. The
synthesis is initiated at multiple primers bound to the template.
However, primers still present in the reaction mixture bind
to the displaced strand and are used as additional
initiation points for DNA synthesis. The multiple products
are long concatemeric molecules of double-stranded DNA.
Rolling-circle amplification of viral DNA genomes using phi29 polymerase.
Reimar Johne at al., Cell, 2009
https://www.youtube.com/watch?v=CaFq9cnfTZI
Rolling Cycling Amplification (RCA)
(A)
(B)
Enzymatic activities of phi29 DNA polymerase. The
combination of 50 -to- 30 polymerase activity (A) and strand
displacement activity makes this enzyme suitable for use in
RCA (B). The 30-to-50 ssDNA exonucleolytic activity is
involved in proofreading.
The amplification reaction initiates when the primers anneal to the
template.
When DNA synthesis proceeds to the next starting site, the
polymerase displaces the newly produced DNA strand and continues
its strand elongation.
The strand displacement generates newly synthesized single stranded
DNA template for more primers to anneal.
Rolling Cycling Amplification (RCA)
Techniques for the single-step amplification of whole genomes have been developed into powerful tools for phylogenetic analyses,
epidemiological studies and studies on genome organization. Recently, the bacteriophage phi29 DNA polymerase has been used for the
efficient amplification of circular DNA viral genomes (plant viral DNA, papillomavirus type 16) without the need of specific primers by a
rolling-circle amplification (RCA) mechanism. Various protocols have been applied for detection of novel viruses, for differentiation between
circular and linear forms of viral genomes and for generation of infectious genomic clones directly from specimens.
Further primer annealing and strand displacement on the newly synthesized template results in a hyper-branched DNA network. The sequence
debranching during amplification results in high yield of the products.
Genome sequenced by RCA
Multiple Displacement Amplification (MDA)
MDA can generate 1–2 µg of DNA from single cell with genome
coverage of up to 99%. Products also have lower error rate and
larger sizes compared to PCR based Taq amplification.
General work flow of MDA:
1. Sample preparation: Samples are collected and diluted in the
appropriate reaction buffer (Ca2+ and Mg2+ free). Cells are lysed with
alkaline buffer.
2. Condition: The MDA reaction with Ф29 polymerase is carried out
at 30°C. The reaction usually takes about 2.5–3 hours.
3. End of reaction: Inactivate enzymes at 65°C before collection of the
amplified DNA products
4.DNA products can be purified with commercial purification kit.
*To separate the DNA branching network, S1 nucleases are used to
cleave the fragments at displacement sites. The nicks on the
resulting DNA fragments are repaired by DNA polymerase I.
MDA applications
MDA generates sufficient yield of DNA products. It is a powerful tool of amplifying DNA molecules from
samples, such as uncultured microorganism or single cells to the amount that would be sufficient
for sequencing studies. The MDA products from a single cell have also been successfully used in arraycomparative genomic hybridization experiments, which usually require a relatively large amount of amplified
DNA.
Genome sequencing of single sperm cell have been reported and successfully amplified in preimplantation
genetic diagnosis (PGD) or parental diagnosis. This ensures that an oocyte or early-stage embryo has no
symptoms of disease before implantation.
Sequencing genome of single uncultured cell bacteria cell, such as Prochlorococcus, and single spore of
fungi has been reported.
The success of more MDA based genome sequencing from a single cell provides a powerful tool of
studying diseases that have heterogeneous properties, such as cancer.
Its high fidelity also makes it reliable to be used in the single-nucleotide polymorphism (SNP) allele
detection. Due to its strand displacement during amplification, the amplified DNA has sufficient coverage of
the source DNA molecules, which provides high quality product for genomic analysis.
Polony Sequencing
PCR
5’
3’
PCR
3’
3’
5’
Brigge PCR and sequencing by synthesis
Pacific Biosciences (Zero Mode waveguides)
1.
A DNA polymerase is immobilized onto a surface
2.
Single strand DNA molecule to be sequenced runs
through the polymerase
3.
Dye-labeled nucleotides are
DNA/polymerase combination
4.
Each time a nucleotides is incorporated, the dye is held
near the surface for a short time
5.
After incorporation, the dye is cleaved away
exposed
to
the
https://www.youtube.com/watch?v=v8p4ph2MAvI
Pacific Biosciences (Single Molecule Real Time Sequencing)
This technique is based on the observation of the performance of polymerase
during DNA synthesis. On this platform, SMRT cells are used, with each cell
having thousands of zero-mode waveguides (ZMWs), which are holes in a
surface that acts as a nanoscale chamber. In each ZMW (which is tens of
nanometers in diameter), a single molecule of DNA polymerase is attached to
the bottom surface.
The accuracy and the speed of performance of the polymerase depend on high
concentrations of nucleotides, and since the nucleotides are fluorescently
labeled, this will lead to the background noise that creates difficulties in
nucleotide incorporation detection. To overcome this problem, the detection
volume in SMRT has been reduced to 20 zeptoliters (10-21 liters). This
considerable reduction of detection volume can reduce the effect of
background noise. One of the main differences between SMRT and previously
described methods is the site of attachment of the fluorescent label. In
other systems, the fluorescent label is attached to the base in nucleotides
and consequently the labels remain attached after nucleotide
incorporation, which leads to an increase in background noise.
Moreover, incorporation of multiple bases will also lead to the creation of a
steric hindrance as a result of the physical bulk of several dye molecules,
which in turn leads to the limitation of enzyme activity. In SMRT technology,
the fluorescent label is attached to the phosphate chain, and as a result
of nucleotide incorporation the pentaphosphate-label couple will be
removed from the nucleotides and will diffuse out of the reaction volume.
Single-cell genome sequencing
Single-cell RNA sequencing workflow
Single-cell exome sequencing
Single-cell exome sequencing reveals
single-nucleotide mutation characteristics of a kidney tumor
https://www.youtube.com/watch?v=Fz7q__qol9g
https://www.youtube.com/watch?v=C8RNvWu7pAw
https://www.youtube.com/watch?v=O4bBZ_UOcK8
NGS Applications
Single-cell applications
Fixed cell
FISH application: species identification
Biofilm: aggregazione complessa di microrganismi contraddistinta
dalla secrezione di una matrice adesiva e protettiva
FISH is often used in clinical studies. If a patient is infected with a
suspected pathogen, bacteria, from the patient's tissues or fluids,
are typically grown on agar to determine the identity of the
pathogen. Many bacteria, however, even well-known species, do
not grow well under laboratory conditions. FISH can be used to
detect directly the presence of the suspect on small samples of
patient's tissue.
FISH can also be used to compare the genomes of two biological
species, to deduce evolutionary relationships. A similar
hybridization technique is called a zoo blot. Bacterial FISH probes
are often primers for the 16s rRNA region.
FISH is widely used in the field of microbial ecology, to identify
microorganisms. Biofilms, for example, are composed of complex
(often) multi-species bacterial organizations. Preparing DNA probes
for one species and performing FISH with this probe allows one to
visualize the distribution of this specific species within the biofilm.
Preparing probes (in two different colors) for two species allows to
visualize/study co-localization of these two species in the biofilm,
and can be useful in determining the fine architecture of the biofilm.
FISH application: medical diagnosis
Often parents of children with a developmental disability want to know more about their child's conditions before choosing to have another child.
Examples of diseases that are diagnosed using FISH include Prader-Willi syndrome, Angelman syndrome, 22q13 deletion syndrome, chronic
myelogenous leukemia, acute lymphoblastic leukemia, Cri-du-chat, Velocardiofacial syndrome, and Down syndrome.
FISH on sperm cells is indicated for men with an abnormal somatic or meiotic karyotype.
In medicine, FISH can be used to form a diagnosis, to evaluate prognosis, or to evaluate remission of a disease, such as cancer. FISH can also
be used to detect diseased cells more easily than standard cytogenetic methods. FISH does not require living cells and can be
quantified automatically, a computer counts the fluorescent dots present. However, a trained technologist is required to distinguish subtle
differences in banding patterns on bent and twisted metaphase chromosomes. FISH can be incorporated into Lab-on-a-chip microfluidic device.
This technology is still in a developmental stage but, like other lab on a chip methods, it may lead to more portable diagnostic techniques.
The different steps of the karyotyping procedure: Giemsa staining
Fluorescence in situ hybridization (FISH)
FISH is a cytogenetic technique that uses fluorescent probes that bind to only those parts of the chromosome with a high degree of sequence
complementarity. It was developed by biomedical researchers in the early 1980s and is used for detecting RNA (mRNA, long non-coding RNA and miRNA) or DNA
sequences in the cells, tissues, and tumors and localize the presence or absence of specific DNA sequences on chromosomes.
This is a technique in which single-stranded nucleic acids (usually DNA, but RNA may also be used) are permitted to interact so that complexes, or
hybrids, are formed by molecules with sufficiently similar, complementary sequences. Through nucleic acid hybridization, the degree of sequence identity can
be determined, and specific sequences can be detected and located on a given chromosome.
The differences between the various FISH techniques are usually due to variations in the sequence and labeling of the probes; and how they are used in
combination. Probes are divided into two generic categories: cellular and acellular.
FISH
FISH has a large number of applications in molecular biology and medical science, including gene mapping, diagnosis of chromosomal
abnormalities, and studies of cellular structure and function. Chromosomes in three-dimensionally preserved nuclei can be "painted" using
FISH. In clinical research, FISH can be used for prenatal diagnosis of inherited chromosomal aberrations, postnatal diagnosis of carriers of
genetic disease, diagnosis of infectious disease, viral and bacterial disease, tumor cytogenetic diagnosis, and detection of aberrant gene
expression. In laboratory research, FISH can be used for mapping chromosomal genes, to study the evolution of genomes (Zoo FISH), analyzing
nuclear organization, visualization of chromosomal territories and chromatin in interphase cells, to analyze dynamic nuclear processes,
somatic hybrid cells, replication, chromosome sorting, and to study tumor biology. It can also be used in developmental biology to study the
temporal expression of genes during differentiation and development. Recently, high resolution FISH has become a popular method for ordering
genes or DNA markers within chromosomal regions of interest.
Hybridization process – DNA
 The probe must be large enough to hybridize
specifically with its target. The probe is tagged directly
with fluorophores, with targets for antibodies or with
biotin. Tagging can be done in various ways, such as
nick translation, or PCR using tagged nucleotides.
 The probe is then applied to DNA and incubated for
approximately 12 hours while hybridizing. Several wash
steps remove all unhybridized or partially hybridized
probes. The results are then visualized and quantified
using a microscope that is capable of exciting the dye
and recording images.
 Fluorescent signal strength depends on many factors
such as probe labeling efficiency, the type of probe,
and the type of dye. Fluorescently tagged antibodies or
streptavidin are bound to the dye molecule. These
secondary components are selected so that they have
a strong signal.
Hybridization process – DNA
Hybridization process – DNA
A) Direct FISH detection.
Fluorescent
labels
are
attached to a probe which will
hybridize to a target DNA
strand.
B) Indirect FISH detection. Biotin, for example,
is attached to a probe. Streptavidin linked to a
fluorescent tag binds biotin with high specificity.
Chromogenic in situ hybridization (CISH) is a
cytogenetic technique that combines the
chromogenic signal detection method of
immunohistochemistry (IHC) techniques with in
situ hybridization.
Probe design for CISH is very similar to that for
FISH with differences only in labelling and
detection. FISH probes are generally labelled
with a variety of different fluorescent tags and
can only be detected under a fluorescence
microscope, whereas CISH probes are
labelled with biotin or digoxigenin
C) Indirect CISH detection. Again, biotin is
attached to a probe. Streptavidin linked to
horseradish peroxidase binds biotin with
high specificity. Horseradish peroxidase
(HRP) converts diaminobenzidine into a
brown precipitate.
Variations on probes and analysis
 Probe size is important because longer probes hybridize less specifically than
shorter probes, "a short strand of DNA or RNA (often 10–25 nucleotides)
which is complementary to a given target sequence, it can be used to identify
or locate the target: if the goal of an experiment is to detect the breakpoint of
a translocation, then the overlap of the probes defines the minimum window in
which the breakpoint may be detected.
 Probes that hybridize along an entire chromosome are used to count the
number of a certain chromosome, show translocations, or identify extrachromosomal fragments of chromatin. This is often called "wholechromosome painting“. However, it is possible to create a mixture of smaller
probes that are specific to a particular region (locus) of DNA; these mixtures
are used to detect deletion mutations. When combined with a specific color,
a locus-specific probe mixture is used to detect very specific
translocations.
 A variety of other techniques use mixtures of differently colored probes. the
probe mixture for the secondary colors is created by mixing the correct ratio of
two sets of differently colored probes for the same chromosome. This
technique is sometimes called M-FISH. The same physics that make a variety
of colors possible for M-FISH can be used for the detection of translocations.
An example is the detection of BCR/ABL translocations (the Philadelphia
chromosome or Philadelphia translocation is a specific abnormality of
chromosome 22, which is unusually short, as an acquired abnormality that is
most commonly associated with chronic myelogenous leukemia).
Multicolour fluorescence in situ hybridisation (M-FISH)
Multicolour fluorescence in situ hybridisation (M-FISH) and multicolour
banding (M-BAND) are advanced chromosome painting techniques
combining multiple chromosome- or region-specific paints in one step.
M-FISH identifies all chromosomes or chromosome arms at once,
whereas M-BAND identifies the different regions of a single
chromosome.
The use of either or both can improve the accuracy of karyotyping and help
identify cryptic chromosome rearrangements.
These probes are prepared by pooling multiple chromosome- or
chromosome region-specific DNA libraries, each labelled with a unique
combination of fluorochromes. In the protocol described here, a
commercial probe is used. Well-spread metaphases are prepared
according to standard techniques, followed by alkaline denaturation and
application of the denatured probe. After an incubation period, the slides
are washed. A fluorescence microscope with filter sets specific to the
fluorescent labels is used for analysis, together with specialized image
analysis software. The software interprets the combination of
fluorochromes to identify each chromosome.
Methods Mol Biol. 2011;730:203-18.
The use of M-FISH and M-BAND to define chromosome abnormalities. Mackinnon RN, Chudoba I.
Spectral karyotyping to study chromosome abnormalities
and chromosome painting
Spectral karyotyping is an image of
colored
chromosomes.
Spectral
karyotyping involves FISH using
multiple forms of many types of
probes with the result to see each
chromosome labeled through its
metaphase stage.
This type of karyotyping is used
specifically when seeking out
chromosome arrangements.
multicolour banding (M-BAND)
Hybridization process – RNA
A target-specific probe, composed of 20 oligonucleotide pairs, hybridizes to the target RNA(s). Separate but
compatible signal amplification systems enable the multiplex assay (up to two targets per assay). Signal
amplification is achieved via a series of sequential hybridization steps. At the end of the assay the tissue
samples are visualized under a fluorescence microscope.
RNA detection in fixed probes
A set of Stellaris FISH probes
comprises multiple oligonucleotide
probes
each
labeled
with
a
fluorophore that bind to targeted
transcripts. This technology was
developed by Arjun Raj/UMDNJ and
was formerly known as Single
Molecule FISH, which is now
commercially available as Stellaris
FISH Probes
3,5-difluoro-4-hydroxybenzylidene
imidazolinone (DFHBI)
And Spinach is a synthetically derived
RNA
aptamer
created
using
Systematic Evolution for Ligands
Stellaris(R)
MS2 tagging is a technique based
upon the natural interaction of the
MS2 bacteriophage coat protein with
a stem-loop structure from the phage
genome
The NanoFlare contains a monolayer
of
antisense
DNA (recognition
sequence) adsorbed to the surface of
a 13-nm spherical gold nanoparticle. A
reporter flare sequence is hybridized
to the recognition sequence, which
contains a fluorophore (red). The dye
is quenched in close proximity to the
gold surface. The reporter flare is
displaced
when
complementary
mRNA (blue) binds the recognition
sequence, providing a fluorescent
signal
Stellaris(R) RNA FISH probes
Stellaris RNA FISH, formerly known as Single Molecule RNA FISH, is a method of detecting and quantifying mRNA and other long RNA molecules in a thin layer of
tissue sample. Targets can be reliably imaged through the application of multiple short singly labeled oligonucleotide probes. The binding of up to 48 fluorescent
labeled oligos to a single molecule of mRNA provides sufficient fluorescence to accurately detect and localize each target mRNA in a wide-field fluorescent
microscopy image. Probes not binding to the intended sequence do not achieve sufficient localized fluorescence to be distinguished from background.
Single-molecule RNA FISH assays can be performed in simplex or multiplex, and can be used as a follow-up experiment to quantitative PCR, or imaged simultaneously
with a fluorescent antibody assay. The technology has potential applications in cancer diagnosis, neuroscience, gene expression analysis, and companion diagnostics.
Stellaris(R) RNA FISH probes
Molecular beacons as FISH probes
MS2 system
MS2 tagging is a technique based upon the natural interaction of the MS2
bacteriophage coat protein with a stem-loop structure and partnered to
GFP for detection of RNA in living cells.
MS2 protein
Spinach system
Nano-flares
A
B
Quantitative Fluorescent in situ hybridization (Q-FISH)
Q-FISH is a cytogenetic technique based on the traditional
FISH methodology. In Q-FISH, the technique uses
labelled (Cy3 or FITC) synthetic DNA mimics called
peptide nucleic acid (PNA) oligonucleotides to quantify
target sequences in chromosomal DNA using fluorescent
microscopy and analysis software. Q-FISH is most
commonly used to study telomere length, which in
vertebrates are repetitive hexameric sequences
(TTAGGG) located at the distal end of chromosomes.
Telomeres are necessary at chromosome ends to prevent
DNA-damage responses as well as genome instability. To
this day, the Q-FISH method continues to be utilized in the
field of telomere research.
Q-FISH is commonly used in cancer research to
measure differences in telomere lengths between
cancerous and non-cancerous cells. Telomere shortening
causes genomic instability and occurs naturally with
advanced age, both factors that correlate with possible
causes of cancer.
Also, unlike Southern blots which need over 105 cells for a
blot, less than 30 cells are needed in Q-FISH.
Q-FISH
Although the quantitative ability of Q-FISH is most commonly
used in telomere research, other fields that only require
qualitative data have adopted the use of PNAs with FISH for
both research and diagnostic purposes. PNA-FISH can be
used to screen blood cultures for various strains of
bacteria. PNA-FISH assays have been developed for
identifying and diagnosing infectious diseases in a rapid
manner within the clinic. Combined with traditional gram
staining of positive blood cultures, PNAs can be used to target
species-specific rRNA (ribosomal RNA) to identify different
strains of bacteria or yeast. Since the test can be performed
relatively quickly, the test is being considered for use in
hospitals where hospital-acquired infections can occur.
Similar to Q-FISH, Flow-FISH is an adaptation of Q-FISH
that combines the use of PNAs with flow cytometry. In this
method, Flow-FISH uses interphase cells rather than
metaphase chromosomes and hybridizes the PNA probes in
suspension. Following hybridization, thousands of cells can be
analyzed on a flow cytometer in a relatively short time.
However, Flow-FISH only provides an average telomeric
length for each cell whereas Q-FISH is able to analyze the
telomere length of an individual chromosome.
Advanced in cell diagnostics: RNAscope is alternative solution to FISH
Microarray and PCR provide useful molecular profiles of diseases, but clinically relevant information regarding cellular and tissue context, as
well as spatial variation of the expression patterns, is lost in the process.
RNAscope® in situ assays provides the first opportunity to profile single cell gene expression in situ, unlocking the full potential of RNA
biomarkers. The targeted molecular signature of every cell in a sample is revealed and measured precisely, all within the intricate cellular
and tissue architecture of clinical specimens.
RNA in situ hybridization using RNAscope® technology is the only platform that has the sensitivity to detect every gene in the
human transcriptome in situ, and is signal amplification methodology and to simultaneously quantify multiple mRNA transcripts at
a single cell level.
RNAscope technology
Double "Z" oligo probes are designed to hybridize to specific RNA target
The double "Z" oligo probes are designed to hybridize to specific RNA target of interest. Each target RNA is designed with 20 Z probe
pairs over a 1 Kb region. We can design probes for virtually ANY gene in ANY genome for interrogation in ANY tissue or Cells.
Double "Z" oligo probes are designed to hybridize to specific RNA target
RNAscope® Probe Design and Signal Amplification Strategy
In order to substantially improve signal-to-noise ratio of RNA ISH, RNAscope® employs a prove design strategy much akin to fluorescence
resonance energy transfer (FRET), in which two independent probes (double Z probes) have to hybridize to the target sequence in tandem in
order for signal amplification to occur. Because it is highly unlikely that two independent probes will hybridize to a nonspecific target
right next to each other, this design concept ensures selective amplification of target-specific signals.
Each Z target probe contains three
elements:
• The lower region of the Z is an 18-to
25-base region that is complementary
to the target RNA. This sequence is
selected for target specific
hybridization and uniform hybridization
properties.
• A spacer sequence that links the two
components of the probe.
• The upper region of the Z is a 14-base
tail sequence. The two tails from a
double Z probe pair forms a 28 base
binding site for the pre-amplifier.
20 Double Z probe pairs
bind to target RNA.
Signal amplification is achieved by a
cascade of hybridization events:
• 20 double Z target probe pairs
hybridize to a 1KB region of the target
RNA
• Preamplifiers bind to the 28 base
binding site formed by each Double Z
probe pairs.
• Amplifiers are then bind to the 20
binding sites on each preamplifier.
• Label probes containing fluorescent
molecule or chromogenic enzyme bind
to the 20 binding sites on each
amplifer.
Preamplifiers hybridize to double Zs,
amplifiers hybridize to preamplifiers and
label probes hybridize to amplifiers creating
strong signals for each RNA molecule.
Advantages RNAscope® probe design and signal amplification strategy
• Sensitivity: Detection of each single RNA molecule requires only three
double Z probe pairs to bind to target RNA. The 20 double Z probe pairs
provide robustness against partial target RNA accessibility or degradation.
• Specificity: The double Z probe design prevents background noise. Single
Z probes binding to nonspecific site will not produce a binding site for the
pre-amplifer, thus preventing amplification of non-specific signals,
contributing to specificity.
• Single molecule visualization and quantitation: The 20x20x20 probe
design and signal amplification increases sensitivity such that a single
molecule of RNA can be visualize as a punctuate signal dot under a standard
microscope.
• Compatible with Degraded RNA: The double Z probe design, with its
relatively short target region (40-50 bases of the lower region of the
double Z) allows for successful hybridization of partially degraded RNA.
Visualize histone modifications in single cells? Yes you can
ChIP has been a dear friend to researchers studying histone
modifications for years. But, it has developed a way to zoom
into specific cell types in tissue to map out the histone
modifications at specific loci: The method, called in situ
hybridization-proximity ligation assay (ISH-PLA), is, you
guessed it, a combination of in situ hybridization and
proximity ligation.
A gene of interest is hybridized with a biotin-tagged DNA
probe (red). Next, an anti-biotin antibody (pink) and an
antibody (blue) recognizing an epigenetic mark (for
example histone H3 methylation) are applied. These
antibodies are then each tagged with PLA (Proximity
Ligation Assay) antibodies (orange and yellow). If the biotin
and epigenetic mark are in close proximity, the two PLA
antibodies will interact and create a signal detectable
with a fluorescent DNA probe.
ISH-PLA: a new method of detection of histone modifications
at a single genomic locus in tissue sections
o Biotinylated probe target the gene of interest
o Another probe target chromatin modification
o 2nd Antibody with PLA
o Rolling circle amplification
o Detection of rolling circle products
This methodology promises applications in the
study of epigenetic mechanisms in complex
multicellular tissues in development and
disease.