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Lab Materials Chemicals Storage CentrifugeWorkstation.

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Presentation on theme: "Lab Materials Chemicals Storage CentrifugeWorkstation."— Presentation transcript:

1 Lab Materials Chemicals Storage CentrifugeWorkstation

2 Procedure for Isolating Polymorphonuclear Cells Blood (Cancer patient) “Buffy Coat” Storage

3 Lab Materials

4 Isolation of RNA Using Immunomagnetic Beads

5 Immunomagnetic Isolation of mRNA

6 Images of Immunomagnetic Isolation of CTC Immunomagnetic Beads Target Ber-Ep4 Epithelial Cell Antigen (ECA)

7 Reverse Transcriptase Polymerase Chain Reaction  Animation Animation

8 Return To Immunology Home Page Return to Biology Home Page

9 Polymerase Chain Reaction (PCR)

10 Lab Materials PCR MachinesIncubator PCR WorkstationCold Room

11 Identifying Type of Cancer Cell  Most cells have three types of genes: 1. Housekeeping Genes (Actin) 2. Tissue-specific Genes (Cytokeratin-19) 3. Organ Specific Genes (Mammaglobin) 4. Cancer Specific Genes (BRCA1 & BRCA 2)

12 Gel Electrophoresis Molecular Markers: 1.Actin (~350 bp) 2.Cytokeratin-19 (~300 bp) 3.Mammaglobin (~280 bp)

13 RT-PCR Results in the Detection of Breast CTC Positive Result: + Negative Result: - Sample #ActinCytokeratin 19MammaglobinOverall 1 ++-+ 2 ++++ 3 +--- 4 +--- 5 +--- 6 ++++ 7 ++++ 8 ++-+ 9 ++-+ 10 +--- 11 ++++ 12 +--- 13 ++-+ 14 ++-+ 15 ++-+ Percentage100%67%27%67%

14 Conclusion  Use of multiple markers enhances sensitivity  Cancer cells can be detected at early stages  Less radiation therapy, chemotherapy, and immunotherapy

15 Future Plans  Use additional molecular markers to enhance sensitivity (Telomerase)  Associate risk factors genes (BRCA1 & BRCA2)

16 References  Cotran, Kumar, Collins: The Pathological Basis of Disease 6th Edition (W.B. Saunders Company)  Hospital Practice (http://www.hosppract.com/)http://www.hosppract.com/  Foss AJ, Guille MJ, Occleston NL, Hykin PG, Hungerford JL, and LightmanS: The detection of melanoma cells in peripheral blood by reverse transcriptase polymerase chain reaction. Br J Cancer 72: 155 – 159, 1995  Ghossein RA, Bhattacharya S, Coit DG. Reverse transcriptase polymerase chain reaction (RT-PCR) detection of melanoma-related transcripts in the peripheral blood and bone marrow of patients with malignant melanoma. What have we learned? Recent Results Cancer Res. 2001;158:63-77. Ghossein RA, Bhattacharya S, Coit DG.  Ghossein RA, Bhattacharya S.: Molecular detection and characterization of circulating tumour cells and micrometastases in solid tumours. Eur J Cancer. 2000 Aug 36 (13 Spec No):1681-94.  NATIONAL BREAST CANCER FOUNDATION (http://www.nationalbreastcancer.org/)http://www.nationalbreastcancer.org/  Pelkey TJ, Frierson HF and Bruns DE: Molecular and immunological detection of circulating tumor cells and micrometastases from solid tumors. Clin Chem 42:1369 – 1381, 1996  Ronald A. Ghossien, Leo Carusone, and Satyajit Bhattacharya: Molecular detection of micrometastases and circulating tumor cells in melanoma prostatic and breast carcinomas. In vivo 14: 237 – 250, 2000

17 Acknowledgements  Dr. Sat Bhattacharya Memorial Sloan- Kettering Cancer Research Center Harlem Children Society Minimal Residual Lab (MRD Lab)

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19 Thank You for Your Cooperation! Don’t be afraid to ask questions Rami Pankaj


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