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in-situ PCR

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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.

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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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

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

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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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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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

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

Applications

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• Widely used in areas such as

• Embryogenesis

• Organogenesis

• Infectious diseases

• Genetics

• Neoplasia or pathology

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

In Situ PCR

•  Supersensi1ve:ISP1-2copies/cellvsCISH>10copies/cell

•  Producereliableandreproducibleresults

•  Veryspecific:nostaininginthenega1vecontrolsamples.

•  Fullyautomated:canbefinishedbyXmatrx

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