BASIC INVESTIGATIVE

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BASIC INVESTIGATIVE TECHNIQUES IN EXPERIMENTAL
PATHOLOGY
Adolfo J. de Bold
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SYNOPSIS A RESEARCH ADVENTURE INTO THE CARDIAC
PHENOTYPE LEADS TO THE DISCOVERY OF THE ENDOCRINE
FUNCTION OF THE HEART BY MEANS OF AN
INVESTIGATIVE APPROACH EMPLOYING VARIOUS
MORPHOLOGICAL TECHNIQUES
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BACKGROUND I AS EARLY AS THE 19TH CENTURY LIGHT
MICROSCOPY OBSERVATIONS OF THE HEART HAD
DETECTED PHENOTYPIC HETEROGENEITY AT THE CELLULAR
LEVEL. T HAT IS, DIFFERENT CELL TYPES WERE
RECOGNIZED AND ASSOCIATED WITH SOME BASIC
PROPERTIES OF ALL CELLS INCLUDING CONTRACTION
(WORKING CARDIOCYTES), CONDUCTION (i.e. CELLS OF
THE PURKINJE SYSTEM) AND EXCITATION (e.g. CELLS
IN THE SINO-ATRIAL NODE)
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BACKGROUND II HOWEVER, THE INTRODUCTION OF THE
ELECTRON MICROSCOPE AND ITS USE TO STUDY
CARDIOCYTES WITH MUCH MORE DETAIL (RESOLUTION)
DEMONSTRATED THAT THE CARDIOCYTES OF THE ATRIA
HAVE STORAGE GRANULES - REFERRED TO AS SPECIFIC
ATRIAL GRANULES - REMINISCENT OF GRANULES KNOWN
TO BE USED BY THE CELL TO STORE PRODUCTS FOR
EXPORT (e.g. HORMONES, ENZYMES). OUR ADVENTURE
BEGINS BY ASKING WHAT IS THE BIOLOGICAL
SIGNIFICANCE OF THIS APPARENT MIXED
(CONTRACTILE/ SECRETORY) PHENOTYPE?
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TRANSMISSION ELECTRON MICROSCOPY OF VENTRICULAR
CARDIOCYTE
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TRANSMISSION ELECTRON MICROSCOPY OF ATRIAL
CARDIOCYTE
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ATRIAL GRANULE
GOLGI COMPLEX
HIGH POWER VIEW OF SECRETORY COMPLEX IN AN ATRIAL
CARDIOCYTE
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BACKGROUND III AS IT HAPPENS IN ANY BRANCH OF
SCIENCE, THE PROCESS LEADING TO AN EXPLANATION
OF A PHENOMENON BEGINS WITH THE FORMULATION OF
AN HYPOTHESIS BASED ON AN EXTENSION OF PAST
KNOWLEDGE. IN THE CASE OF THE SPECIFIC ATRIAL
GRANULES IT WAS HYPOTHESIZED THAT THESE GRANULES
CONTAINED A PRODUCT FOR EXPORT BECAUSE OF THE
STRUCTURAL SIMILARITIES OF THESE GRANULES AND
ASSOCIATED COMPONENTS (e.g. GOLGI COMPLEX) TO
THOSE FOUND IN ENDOCRINE AND EXOCRINE CELLS.
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BACKGROUND IV IN ADDITION, SOME DATA SUGGESTED
THAT THE SPECIFIC ATRIAL GRANULES WERE A SITE FOR
STORAGE OF CATECHOLAMINES. MORE SPECIFICALLY,
NOREPINEPHRINE. GIVEN THAT HISTOCHEMICAL AND
BIOCHEMICAL APPROACHES TO SETTLE THIS QUESTION
GAVE CONFLICTING RESULTS, IT WAS DECIDED THAT THE
ACCEPTANCE OR REJECTION OF THE WORKING
HYPOTHESIS WAS TO ISOLATE THE SPECIFIC ATRIAL
GRANULES FROM ATRIAL MUSCLE FOLLOWED BY CHEMICAL
DETERMINATION OF THEIR CATECHOLAMINE CONTENT.
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BACKGROUND V THE INVESTIGATIVE TECHNIQUE CHOSEN
TO ISOLATE THE GRANULES IS WIDELY USED FOR THE
STUDY OF CELLS AND TISSUES BECAUSE IT IS THE
MOST EFFECTIVE MEANS TO ISOLATE FRACTIONS
ENRICHED IN SPECIFIC CELLULAR COMPONENTS. THE
SEPARATION IS BASED IN THE BEHAVIOR OF THESE
COMPONENTS IN A GRAVITATIONAL FIELD GIVEN THEIR
SIZE AND DENSITY. THIS INVESTIGATIVE TECHNIQUE
IS REFERRED TO AS TISSUE FRACTIONATION USING
DIFFERENTIAL AND DENSITY GRADIENT CENTRIFUGATIONS
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Each class of organelle (nuclei, mitochondria,
lysosomes, etc.) has physical characteristics
(size, shape, density, etc) which makes it
different from other classes. Hence, if the
cell is appropriately broken open, its
organelles can be subsequently isolated based on
their physical properties using centrifugation .
The process of breaking open cells is called
homogenization. Homogenization of mammalian
cells or tissues is often carried out in
solutions containing agents to preserve pH and
osmotic pressure (e.g. 0.25 M sucrose, pH 7.2),
a chelating agent (EDTA), a high molecular weight
polymer to reduce organelles interactions
(glycogen, albumin) and a high salt concentration
to help solubilize proteins (KCl). The actual
breakage of the tissue and cells or
homogenization is accomplished by rough tissue
chopping followed by the use of devices such as
the Polytron (mechanical plus ultrasonic forces)
and/or a tube and pestle arrangement such as a
the Potter-Elvehjem homogenizer (shear forces).

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The subsequent isolation of organelles from
homogenates is called fractionation. The
commonest way to carry out subcellular
fractionation is by centrifugation. The
employment of differential centrifugation to
prepare crude fractions of subcellular particles
from homogenates is often a necessary step prior
to a subsequent further purification of one or
more particles by density gradient
centrifugation. Differential centrifugation is
typically carried out using angle rotors in
high speed centrifuges (lt20,000 rpm gt20,000 rpm
ultracentrifuge). Stepwise increase of speed
using these rotors gives rise to fractions with
names that are more or less standard regardless
of the tissue being used as follows (rcf 1.12
x 10-5 x Tip Radius in cm x rpm2 ) The Nuclear
Pellet contains, in addition to nuclei,
mitochondria, sheets of plasma membrane and, if
the homogenate has not been filtered, unbroken
cells and debris (including connective tissue).
This pellet is obtained when the homogenate is
centrifuged 5000 -10000 g-min (e.g. 500 1000 g
x 10 minutes).
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The Heavy Mitochondrial Pellet contains
predominantly, mitochondria with rather few
contaminants. Minor components such as lysosomes,
peroxisomes, Golgi membranes and various
membrane vesicles are present largely because of
entrapment during the pelleting process (1,000
3,000 g x 10 min). The Light Mitochondrial
Pellet (in our case Crude Granule Fraction)
contains mitochondria, lysosomes, peroxisomes,
secretory granules Golgi membranes and some
endoplasmic reticulum. Of all differential
centrifugation fractions it is the most variable
in terms of the actual centrifugation parameters
used g-forces of 15-20,000g and times of 10-20
min are the most common. The Microsomal Pellet
is rather better defined and contains only
membrane vesicles. Some of those vesicles will
have been present in the cell before
homogenization (e.g. endosomes, secretory
vesicles and vesicles from the trans-Golgi
network), others from the plasma membrane, Golgi
and smooth and rough endoplasmic reticulum, will
have been produced by the homogenization
procedure (15.104 x 60 -120 min). The High
Speed Supernatant is what remains of the
homogenate after the microsomal pellet is
obtained. No particulated elements are present
in this fraction. Only soluble cytoplasmic
components are found (e.g. enzymes).
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Density gradient centrifugation is often used to
further fractionate a fraction of the
homogenate as obtained during differential
centrifugation. Sucrose gradients (0.25 2.2 M)
formed in centrifugation tubes is the
classical separation medium. Continuous
gradients are used for analytical purposes while
discontinuous gradients are used for preparative
purposes. Other media that do not have some of
the disadvantages of sucrose are also used such
as Percoll and OptiPrep. Gradients can be
formed using pumps configure to mix two solutions
of different densities in a linear or non-linear
mode or can be obtained by layering by hand
solutions of different densities or, in the case
of high molecular weight media, they can be
self-forming during centrifugation. Centrifuge
rotors can be fixed angle or swinging out
bucket. After the appropriate centrifugation time
(e.g. 2-10.105 g x 60- 90 min) , subcellular
organelles distribute in bands (fractions)
throughout the gradient. The fractions are
recovered either by aspiration, piercing the
bottom of the tube or slicing it. The
distribution of organelles in the gradient can be
determined by measuring marker enzymes or other
components of the organelles.
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A band of subcellular organelles is seen after
density gradient centrifugation
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Examples of markers
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MICROSOMAL
CRUDE GRANULAR
MITOCHONDRIAL
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Subcellular distribution of activities after
density gradient centrifugation of a
post-nuclear atrial homogenate fraction. Shown
are ANF (Panel A), Go? by Western blot and by
densitometry reading relative to protein (panels
B and C respectively) and the marker enzymes
glucose-6-phosphatase (endoplasmic reticulum)
succinate dehydrogenase (mitochondria) and
galactosyl transferase (Golgi).
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The information obtained using a discontinuous
density gradient to isolate the specific atrial
granules showed that these organelles do not
contain appreciable amounts of catecholamine
thus not supporting the original hypothesis.
Hence alternative approaches were necessary to
try to elucidate the nature and function of the
atrial granules. It was decided to take this
problem from the electron microscopic level to
the light microscopic level (why?). A series of
histochemical and staining techniques were
applied to sections of atrial tissue in order to
get an insight into the composition of the
specific atrial granules. These included
aldehyde fuchsin after thiosulfation,
lead hematoxylin, diaminobenzidine reaction for
indole and others designed to detect
carbohydrates or lipids. In addition, in order
to compare the kinetics of newly synthesized
protein handling by the Golgi complex/specific
atrial granule system to that of secretory
cells, studies were carried using tritiated
leucine and autoradiography. Lets first look
at the studies using labeled amino acids to
detect newly synthesized protein. The technique
is of general application for the detection of
radiolabeled molecules at the ultrastructural
level.
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Light and electron microscopic autoradiography in
the study of synthesis and vectorial transport
of proteins. The technical strategy to carry out
this technique has three important aspects. 1.
Fixation type. Fixation of the tissue has to
comply with the important condition that the
fixative has to preserve cellular structure and
the compound of interest (radiolabeled protein)
but must not fix the radiolabeled precursor
radiolabeled amino acid (Discuss common fixatives
and their properties). Further, processing of
the tissue for embedding and sectioning must no
wash away the compound of interest. 1.a.
Experiment to determine the degree of non
specific binding of precursor by the
fixative. The simplest experiment consists of
measuring the radioactivity of tissues exposed
to the radioactive amino acid but pre-treated
with the protein synthesis inhibitor
puromycin.
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Radioactivity (cpm . 10-3)
Inhibition of protein synthesis inhibits
incorporation of 3H-Leu into the tissue (i.e.
radioactivity is not due to non-specific binding
of the radiolabelled amino acid)
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1.b. Experiment to determine the loss of
radiolabeled protein during processing for
microscopy. By assaying the different processing
chemicals for TCA-precipitable activity (mainly
fixative and washing solutions) one can asses the
amount of radiolabeled protein loss (Why?)



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2. Duration of the exposure (pulse) of the
tissue to the radioactive precursor. The
duration of exposure of the tissue to the labeled
amino acid must be shorter than the transit of
the labeled protein from one cell compartment to
another (Why?) 3. Resolution and isotope
energy. Fine grain photographic emulsions for
electron microscopy autoradiography have silver
halide grain around 1000 ?. An isotope with low
radiating energy such as tritium will only
interact with neighboring grains while a high
energy particle such as that from 14C or 35S can
travel relatively long distances to interact
with grains away from the source thus reducing
resolution.
EMULSION
SECTION
ORGANELLE
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SUMMARY OF AUTORADIOGRAPHIC PROCEDURE Prepare
Parlodion solution ? coat slides ? assess
thickness ? deposit sections on film ? evaporate
carbon layer ? coat with photographic emulsion ?
expose at 4 oC for appropriate period (weeks)
? develop and fix ? stain sections (L/M) or float
off and pick up on grids and stain (E/M)
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Atrial granule
Golgi
Silver grains of photographic emulsion reveal
sites of 3H-leu incorporation
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CONCLUSION FROM AUTORADIOGRAPHIC EXPERIMENTS
Unlike ventricular cardiocytes, atrial
cardiocytes carry out synthesis and vectorial
transport of a protein with apparent high
turnover in a fashion similar to secretory
cells. O.K. but what protein? How about a hint?
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Atrial granules
ALDEHYDE FUCHSIN AFTER THIOSULFATION (-SH -S-S-)
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Atrial granules
Lead hematoxylin (side chain carboxyl groups)
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SPECIFICITY OF STAIN TESTED IN VENTRICULAR MUSCLE
(NO GRANULES)
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Formaldehyde-HCl in gas phase (indole)
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CONCLUSIONS REACHED SO FAR REGARDING THE NATURE
OF THE SPECIFIC GRANULES CONTENT AS EVIDENCED BY
BIOCHEMICAL, HISTOLOGICAL, AUTORADIOGRAPHIC,
STAINING AND HISTOCHEMICAL EVIDENCE
The specific atrial granules are not a storage
site for catecholamines but contain a
polypeptide with high turnover and high density
of side chain carboxyl groups. This polypeptide
contains sulphur and indole amino acids. O.K.
but what is the function of this
polypeptide? The answer, or at least a
hypothesis regarding function, was made
possible by combining the specific stain
developed for the specific atrial granules
lead hematoxylin, and a powerful technique in
investigative morphology
STEREOLOGY
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Stereology is a set of methods for quantitation
of three-dimensional microscopic structures from
(two dimensional) sections. One simple
application is the determination of relative
volumes or areas using a set of reference
points. It can be demonstrated that when
superimposing a regular grid of reference points
onto a section field, the number of points that
fall on a structure relative to the total is
proportional to the cross sectional area and to
the volume that such structure occupies in the
tissue. Strictly Vobj ap ?
Pobjd Where Vobj volume of the object ap
area associated with each point ? Pobj  sum
of all points counted in all sections. d
distance between two consecutive sections used
to count points.
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If one is interested only in relative measures
(e.g. test vs. control) and absolute measures of
volumes are not needed the relationship can be
simplified considerably to Relative volume
Number of points of a reference grid falling on
structure of interest x 100 Total number of
points falling on the total structure In this
and all other cases the measurements (in fact for
any kind of measurement), the measured component
has to be distributed in such manner as to be
representative of the total. Also, the
component has to be distributed randomly and
finally, the measurements are made without bias.
In addition, given that the data will be
subjected to statistical analysis, typically
using tests of significance for small continuous
data, it must be demonstrated that the data
itself is normally distributed. These principles
were applied to the development of a method to
measure the number of specific atrial granules in
rats. The basic premise was that with such
method one could measure changes in the number of
granules (in fact, granulated areas) after the
animals were subjected to different experimental
procedures and hence one could develop a
hypothesis regarding the function of the specific
atrial granules.
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To develop an stereological method to measure the
degree of granulation of atrial cardiocytes
advantage was taken of the fact that there are
age differences in the degree of granulation.
Rats aged 10 or 6 weeks were chosen.
Granulation differences were expected but were
not apparent. The tissues were fixed and embedded
in a water permeable plastic (why?) and the
sections could be stained with lead-hematoxylin.
Pictures were taken (one could use a digitizer
set up too) at 100 x after the areas were chosen
at low magnification (granulation cannot be seen
at low mag) to avoid operator bias. The picture
were projected onto a screen on which a regular
rectangular grid was drawn. The overall sampling
scheme was as follows 2 groups (4 or 6
week-old rats) x 7 rats/group x 4 section/block
x 4 pictures/section 224 pictures
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REFERENCE GRID USED TO QUANTITATE GRANULATED
AREAS IN CARDIOCYTES
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The total variance (error) 156.1403 0.697
224 0.95017 0.09308
0.15499 1.36016 n.a.b.c a.b.c
a.b a Where a number of
groups b number of animals per group c number
of sections per animal n number of pictures per
section This type of analysis is called an
analysis of variance using a nested
classification. It is a very useful form to
analyze the data because you can see which are
the largest contributors to the overall variance
(error) and therefore change the sampling scheme
to reduce the error if necessary.
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In the analysis of variance above it is assumed
that 1. The data follows a normal distribution
(graph plus X2 test) and, 2. The variances are
homogenous i.e. not significantly different from
each other.
Ratio of variances F Var Group I / Var Group
II 0.9780 / 0.9224 1.0603, Pgt0.05
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• Having established that the function of atrial
  granules is that of
• storing hormones (ANF and BNP) that help maintain
  water and electrolyte
• balance, it is useful to determine where this
  hormone is synthesized.
• The site of tissue expression of a product is
  best demonstrated using
• IN SITU
  HYBRIDIZATION (ISH)
• ISH is based on the fact that labeled single
  stranded fragments of
• of DNA or RNA (probes) can be hybridized to
  complementary sequences of
• DNA or RNA present in the cell to form stable
  hybrids.
• Sensitivity of ISH to reveal mRNA depends on
  several parameters
• 1. Ability of the fixation procedure to
  preserve the mRNA of interest
• Type of and specific activity of probe and method
  of detection
• 3. Hybridization conditions

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ISH

• Ability of the fixation procedure to preserve the
  mRNA of interest
• Generally, buffered 10 formalin prepared from
  paraformaldehyde
• followed by freezing or embedding in paraffin or
  plastic resin are
• the most commonly approaches used.
• Type of and specific activity of probe and method
  of detection
• DNA RNA
• dsDNA sscRNA
• Synthetic oligonucleotides
• ssDNA
• Hybrid detection can be accomplished in a number
  of different ways.
• 3H, 14C, 32P, 35S, 125I, biotin, fluorescein,
  luciferase, digoxigenin, etc.
• followed by autoradiography or phosphor imaging.
  Each has its limitations
• in terms of stability or sensitivity.

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ISH
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ISH

• Hybridization conditions
• Hybridization conditions, as in Northern or
  Southern blotting, determines
• the specificity of the hybridization. In turn,
  these conditions depend
• on probe construction, temperature, pH, and
  formamide and salt concentration
• in the hybridization buffer. As in blotting
  analysis, these parameters can
• be varied to change the degree of stringency
  desired.
• Controls
• Emulsion or other detection system control
• Northern blot
• Immunocytochemistry
• Use of different probe complementary to different
  regions of the transcript
• Absorption of the probe with complementary
  sequences
• Hybridization with probes irrelevant containing
  irrelevant sequences
• 7. Pre-treatment with RNase.

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ISH
An example of ISH using PCR-derived riboprobes

• Generation of PCR-derived riboprobe self
  templates
• Four overlapping oligonucleotides corresponding
  to the coding region of the
• gene of interest are synthesized in such a way as
  obtain PCR product
• containing flanking regions with restriction
  sites and T3 or T7 phage
• polymerase promoters. The quantity of the
  internal primers was highly
• limited so that the reaction causes an asymmetric
  single-stranded
• amplification of the two halves of the total
  sequence due to an excess
• of the two flanking primers. In subsequent PCR
  cycles yield a double-
• stranded full length product. This product is
  purified and transcribed
• in the presence of 35S-UTP using T3 or T7
  polymerase to obtain the sense
• and antisense riboprobes, respectively.

T3-RS
RS T7
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ISH
An example of ISH using PCR-derived riboprobes
RIBOPROBE SEQUENCE(5' ?3') (Sense (), antisense
()
Hpa II/ Ava I
ANF-P1
() ACATTAACCCTCACTAAAGGGACCGGTAGAAGATGAGGTCATGC
CTCCGCAGGCCCTGAGCGA
(-) AGAGAGGGAGCTAAGTGCCGCCCCCGCTTCATCGGTCTGCTCGC
TCAGGGCCTGCGGAGG
P2
() CGGCACTTAGCTCCCTCTCTGAGGTGCCTCCCTGGACTGGGGAAGT
CAACCCGTCTCAGA
P3
(-) TAATACGACTCACTATAGGGCCCGAGAGCACCTCCATCTCTCTG
AGACGGGTTGACTTCC
P4
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ANF STORAGE (peptide)
ANF GENE EXPRESSION (mRNA)
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HUMAN CORONARY ARTERIES
A ADVENTITIA M MEDIA I INTIMA
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HUMAN CORONARY ARTERY ADVANCED ATHEROMATOUS
LESION WITH CALCIFICATION
A ADVENTITIA M MEDIA I INTIMA
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NATRIURETIC PEPTIDES AND INFLAMMATION
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NATRIURETIC PEPTIDES AND CARDIAC ALLOGRAFT
REJECTION

Methotrexate
SoluMedrol
OKT3
OKT3
R

R

x
x
ISHLT CLASSIFICATION
-
3
A
0
1
B
-
3
A
0
1
B
1
A
3
A
0
3
A
1
3
A
0
3
A


1
5
0
0
1
5
0
0
1
5
0
0
1
5
0
0
1
5
0
0
1
5
0
0
1
2
0
0
0
0
1
2
0
0
0
0
1
2
0
0
1
2
0
0
9
0
0
9
0
0
9
0
0
9
0
0
6
0
0
6
0
0
BNP (pg/ml)
ANF (pg/ml)
6
0
0
6
0
0
3
0
0
3
0
0
3
0
0
3
0
0
0
0
0
0
WEEK 1
WEEK 2
WEEK 3
WEEK 4
WEEK 7
WEEK 9
WEEK 5
PREOP
T
I
M
E
BNP
BNP
ANF
ANF
Acute cardiac allograft rejection episodes occur
with a specific increase in plasma BNP.
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IL-1ß and TNF- ? Increase BNP Secretion
What cytokines are upregulated in human cardiac
allograft rejection?
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Gene Expression Analysis of Formalin Fixed
Paraffin Embedded Tissue
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Important Technical Advancement

• Formalin Fixed Paraffin Embedded (FFPE) Tissue
• widely available in storage
• in conjunction with clinical data important for
  retrospective clinical studies
• Hence

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Important Technical Advancement

• Combined with genomics, FFPE tissues can be used
  to validate differentially expressed genes
• This will allow for the discovery therapeutic
  targets and possible prognostic indicators

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Methodology
Sample Preparation

• Obtain Samples
• Tissue scrapes of clinical Biopsies
• Laser Capture Microdissection of cell clusters

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Difficulties with FFPE Tissues

• DNA is more stable and is less degraded when
  extracted from FFPE tissues
• RNA is very degraded due to the formalin fixation
  process
• Formalin fixation modifies RNA by the addition of
  mono-methylol groups to the bases

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Difficulties with FFPE Tissues

• Formalin Fixation also creates cross-linkages
  between nucleic acids and proteins
• Fragmented RNA with cross-linkages creates
  problems for reverse transcription and
  quantitation analysis

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Applicable Techniques

• Routine approaches such as
• -Nothern blot analysis
• -RNase protection analysis
• -S1 nuclease analysis
• -in situ hybridization analysis
• Are NOT suitable for small tissue samples
• and for fragmented RNA samples

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Methodology Continued

• Quality Assessment of RNA integrity and
  suitability for
• QRT-PCR
• Use real time PCR to determine the RNA quality
  and quantity (best)
• Use Gel electrophoresis (poor)
• Use Agilent Bioanalyzer (poor)

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Determination of RNA Integrity

• There are commonly used
• methods for assessing the quality of
  RNA
• Gel electrophoresis
• Agilent BioAnalyzer

• These methods provide a reliable and robust
  solution for RNA fromfrozen tissue but not for
  FFPE tissue.

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Methodology Continued

• Choose a reliable microscale RNA extraction
  method
• use proteinase K (serine protease) digestion to
  remove cross-linkages in RNA sample
• incubate tissue extracts at (60 to 70?) to
  reverse methylol additions
• Reverse Transcription of cellular RNA to produce
  high quality cDNA for QRT-PCR

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Quality Assessment Process
AAAAA

• Quantitative PCR based on amplicons located
  100 (3) and 400(5) bases away from the 3
  end of b-actin gene
• Quantity of RNA is calculated based on Ct
  values from each amplicon
• Ratio of 100(3) base over 400(5) base
  quantity used as a metric for RNA quality
  assessment

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Premise of this Assessment

• Two sets of primers are used one that is closer
  to the 3 end (1650-1717) and the other is closer
  to the 5 end (1355-1472)
• The ratio of the relative abundance of the 3
  segment is compared to the 5 segment

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Premise of this Assessment

• If the ratio of 3/5 is 1 then the initial RNA
  is completely intact
• A ratio of 3/5 greater than 1 indicates RNA
  degradation
• For optimal results in downstream applications, a
  ratio less than 20 is preferred

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GeneChip expression array
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GeneChip expression array
The Affymetrix GeneChip X3P Array was designed
specifically for whole-genome expression
profiling of formalin-fixed, paraffin-embedded
(FFPE) samples. The target sequences on the X3P
Arrays are identical to those used for designing
the Human Genome U133 Plus 2.0 Array, for a
total of 47,000 transcripts with 61,000 probe
sets, although the probes on the two types of
arrays are significantly different. As a result
of the modification to the probe selection
criteria, the majority of the probe sets on the
X3P arrays are selected from the 300 bases at the
most 3' end of the transcripts. This is
different from the standard Affymetrix design
strategy which selects probe sets within the
region of 600 bases proximal to the 3' ends.
http//www.affymetrix.com/products/arrays/index.
affx http//www.affymetrix.com/products/arrays/spe
cific/x3p.affx
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REFERENCES There are many references in the web
to the techniques discussed. The specific
references on which the lecture is based is from
work in our laboratory and these are listed below
among other general references. Tissue
Fractionation Graham, J. and D. Rickwood (ed.).
1997. Subcellular Fractionation A Practical
Approach. New York Oxford University Press. C.
De Duve. 1975. Exploring cells with a centrifuge
Science 189 186-194. Studies on the Relationship
between the Catecholamine Distribution in the
Atrium and the Specific Granules Present in
Atrial Muscle Cells. I. Isolation of a
Purified Specific Granule Subtraction. de Bold,
A.J., and Bencosme, S.A. Cardiovas. Res.,
7351-363, 1973. Studies on the Relationship
between the Catecholamine Distribution in the
Atrium and the Specific Granules Present in
Atrial Muscle Cells. II. Studies on the
Sedimentation Pattern of Atrial Noradrenaline and
Adrenaline. de Bold, A.J., and Bencosme, S.A.
Cardiovas. Res., 7364-369, 1973. Participation
of G Proteins in Natriuretic Peptide Hormone
Secretion from Heart Atria. Bensimon, M. Chang,
A.I., Kuroski de Bold, M.L., Ponce, A., Carreras,
D. and de Bold, A.J. Endocrinology 145
5313-5321, 2004 Histochemistry,
Autoradiography Techniques of radioautography for
medical and biological research. T. Nagata. Br J
Med Biol Res 31 185-195, 1998 Autoradiographic
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