in-situ PCR Presented for: Presented by: Date:

in-situ PCR Presented for: Presented by: Date:

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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 BioGenex offers patent reagents, antibodies as well as the Xmatrx instrument to help to do the whole procedure, automatically. 3

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 4

in situ PCR - Workflow SAMPLE PREPARATION Fixation Paraffin Embedding Sectioning and Slide Preparation Deparaffination Rehydration Proteolytic Digestion in situ PCR in situ RT-PCR DNase DIGESTION (OPTIONAL) RT DNA AMPLIFICATION DIRECT INDIRECT IHC ISH 5 VISUALIZATION

in situ PCR - Workflow Poly-HRP anti-mouse IgG Mouse Anti-Fluorescein antibody PCR products with fluorescein label Fluorescein-dUTP 6

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

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. 8

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). 9

Direct in situ PCR Label incorporated directly into the amplified product during PCR Might lead to false positive results Fluorescein Due to non specific incorporation of label Non specific priming by cdna or DNA fragments Labels used: Biotin or Digoxigenin Biotin 10

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 Labelling of PCR product with DIG Probes conjugated to enzymatic labelling are used widely in in situ PCR Michael Mc Pherson % Simon Moller; PCR II edition, The Basics, 2006 11

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

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. 13

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 15

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 16

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

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