In Situ Polymerase Chain Reaction (In situ PCR) is a powerful method that detects minute quantities of rare or single-copy number nucleic acid sequences in frozen or paraffin-embedded cells or tissue sections for the localization of those sequences within the cells. The principle of this method involves tissue fixing (to preserve the cell morphology) and subsequent treatment with proteolytic digestion (to provide access for the PCR reagents to the target DNA). The target sequences are amplified by those reagents and then detected by standard immunocytochemical protocols. In situ PCR combines the sensitivity of PCR or RT-PCR amplification along with the ability to perform morphological analysis on the same sample, and thus it is an attractive tool in diagnostic applications. One of the most prominent applications is the detection of infectious disease agents including HIV-1, HBV, HPV, HHV-6, CMV, and EBV.

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Date:
in-situ PCR

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3. in situ Hybridization - Definition
• in situ PCR is a method in which the
polymerase chain reaction actually takes
place in the cell on a slide, and the
product can be visualized in the same
way as in traditional in situ hybridization.
• Either DNA or RNA can be detected
through the combined examination of
PCR/RT-PCR and Histology
• It has the sensitivity of PCR and the
spatial resolution of in-situ Hybridization
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BioGenex offers patent reagents, antibodies as well as the Xmatrx instrument
to help to do the whole procedure, automatically.

4. in situ PCR - Principle
• In situ PCR refers to the amplification of specific nucleic acid
sequences and subsequent visualization of the PCR products in
tissue sections.
• Sample preparation
• in situ first strand synthesis
• in situ PCR using either labelled or unlabelled probes
• in situ Hybridization or detection
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5. in situ PCR - Workflow
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DNA AMPLIFICATION
VISUALIZATION
SAMPLE PREPARATION
Fixation
Paraffin Embedding
Sectioning and Slide Preparation
Deparaffination
Rehydration
Proteolytic Digestion
in situ PCR in situ RT-PCR
DNase DIGESTION (OPTIONAL)
RT
DIRECT INDIRECT
IHC ISH

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Poly-HRP anti-mouse IgG
Mouse Anti-Fluorescein antibody
PCR products with fluorescein label
Fluorescein-dUTP
in situ PCR - Workflow

7. Sample preparation
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• Fixation:
• Maintains tissue morphology
• Preserves the integrity of nucleic acids
• Either 2-4% paraformaldehyde or 2-3% glutaraldehyde solution is used
• Sectioning:
• A microtome is used to section the fixed tissues of required thickness and
placed on slides
• Tissue sections are deparaffinized in xylene

8. Sample preparation
• Tissue is permeabilized by treatment with Proteinase-K or pepsin
• Recommended conditions are 30 min at 37oC followed by heat
inactivation of 5ug ml-1 proteinase K
• Additional sample treatment methods
• For RT-PCR, DNAse treatment is required to remove DNA
• RNAse ihhibitors added to the sample prevents RNA degradation
• 0.1M Triethanolamine and 0.25 % acetic anhydride can be used to
reduce the static charge on slides
• For working with peroxidases, quenching of endogenous
peroxidases is a must.
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9. in situ first strand cDNA synthesis
• mRNA cannot be used as a template
• Hence an RT-PCR reaction converts mRNA to cDNA
• Either Avian Myeloblastosis Virus (AMV) and Moloney Murine Leukemia
Virus (M-muLV), and rTth, a heat-stable DNA polymerase from Thermus
thermophilus, can be used
• Types of primers used:
• Oligo(dT)12-18 (it binds to the endogenous poly (A)+ tail of mRNA),
• Random hexanucleotides (these bind at any complementary sequence
throughout the length of mRNA),
• Specific oligonucleotides (based on a specific mRNA sequence).
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10. Direct in situ PCR
• Label incorporated directly into the
amplified product during PCR
• Might lead to false positive results
• Due to non specific incorporation
of label
• Non specific priming by cDNA or
DNA fragments
• Labels used: Biotin or Digoxigenin
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Biotin
Fluorescein

11. Indirect in situ PCR
• Amplified product detected by in situ
hybridization using an internal probe
• Sensitivity depends on the method of
probe labelling and detection
• A variety of labels are available
• Radio labels
• Enzymatic labelling
• Probes conjugated to enzymatic labelling
are used widely in in situ PCR
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Labelling of PCR product with DIG
Michael Mc Pherson % Simon Moller; PCR II edition, The Basics, 2006

12. Applications
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• Widely used in areas such as
• Embryogenesis
• Organogenesis
• Infectious diseases
• Genetics
• Neoplasia or pathology

13. Automation: Xmatrx Infinity
• Multi-functional system supports - IHC, ISH and FISH
• Slide processing options – Random (perform template test at any time),
Continuous (access new slides on an on-going basis) and STAT [Short Turn
Around Time]
• Multi-format specimen processing
• Frozen or FFPE Tissues.
• Cell preparations.
• Smears and Fine Needle Aspirates.
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Results
A) Cervical carcinoma showed positive signals after 20 cycles in situ PCR for HPV-16
B) HPV 16 detected by CISH
C) Cervical carcinoma showed positive signals after 20 cycles in situ PCR for HPV-18
D) HPV 18 detected by CISH

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Negative Control
In situ PCR (HPV16) 20 cycles
Tissue type: N. Breast
Magnification: 100x
In situ PCR (HPV16) 20 cycles
Tissue type: Ca. Colon
Magnification: 100x

17. In Situ PCR
• Super sensi1ve: ISP 1-2 copies/cell vs CISH > 10 copies/ cell
• Produce reliable and reproducible results
• Very specific: no staining in the nega1ve control samples.
• Fully automated: can be finished by Xmatrx

18. Please visit www.biogenex.com for more details on our product por8olio