Wael ElRifai and Sakari Knuutila 1 Introduction 1
A vast amount of genome sequencing data has become available over the past few years and methods to facilitate high-throughput analysis of large sets of genes and samples have been developed to localize novel genes related to human cancer. As advanced robotic applications have made it possible to manufacture high-precision microarrays on glass or membranes, pioneering scientists have introduced several variants of the array technology oligonucleotide arrays 1 , DNA microarrays CGH arrays 2 ,...
Wael ElRifai and Sakari Knuutila 1 Introduction
Screening for chromosomal changes in solid tumors was long hindered by methodological problems encountered in standard cytogenetic analysis. Comparative genomic hybridization CGH , a technique that emerged in 1992 1 has proved to be a powerful tool for molecular cytogenetic analysis of neoplasms. The main prerequisite of the technique is DNA isolated from tumor samples. As no cell culture of tumor material is required, the technique has been successfully used to study fresh and frozen tissue...
Sam Thiagalingam 1 Introduction
The signaling pathways mediated by the transforming growth factor-P TGF-P family of factors are implicated in a wide array of biological processes including cell differentiation and proliferation, determination of cell fate during embryogenesis, cell adhesion and cell death. The recent discovery of the SMAD family of signal transducer proteins as mediators of TGF-P relaying signals from cell membrane to nucleus has revolutionized the understanding of the molecular basis of these processes 1,2 ....
CGH Procedure
For optimum CGH hybridization, use slides aged for 10-20 d at room 3.4.1. Pretreatment and Denaturation see Note 5 1. Refix the slides in methanol acetic 3 1 overnight at 4 C. 2. Incubate the slides in 2X SSC at 42 C for 40 min, then wash in distilled water. 3. Dehydrate the slides in ethanol solution 70 , 85 , and absolute for 5 min each. 4. Denaturate, the maximum of four slides at one time, in prewarmed 65-70 C 70 formamide 2X SSC for 2 min. 5. Quickly, remove slides and dehydrate them in a...
Isolation of Genomic DNA from Peripheral Blood Leukocytes
DNA isolation is performed using the Puregene kit. 1. Add 300 L whole blood to a tube containing 900 L of red blood cell lysis solution. Invert to mix, wait 5 min, then invert again and wait another 5 min. 2. Centrifuge at maximum speed in a microfuge remove the supernatant leaving the visible white cell pellet and 10-20 L of residual liquid. Cap the tube and vigorously vortex to resuspend the cells. 3. Add 300 L cell lysis solution to the tube and pipet up and down to lyse the cells. Incubate...
Gel Electrophoresis Vgy
1. Agarose NuSieve, Bioproducts, Rockland, ME . 2. Tris-borate TBE buffer 0.09 M Tris-borate, 0.002 M EDTA, pH 8.0. 3. Ethidium bromide EtBr solution in water 10 mg mL . 4. Gel-loading buffer 0.05 bromphenol blue, 0.05 xylene cyanole FF, 30 glycerol in water. 5. X174 DNA Hae III marker Promega, Madison, WI . 6. Gel electrophoresis tank, accessories, and power supply. LKB1 Gene PCR Amplification Oligonucleotide Primers LKB1 Gene PCR Amplification Oligonucleotide Primers aF forward primers, R...
DNA Extraction Riz
1. Cut one 5 i and several 10 i sections from the paraffin-embedded normal and tumor tissue blocks with a microtome see Note 2 . Sections from a single block may suffice for DNA extraction if the tissue in that block contains both normal and tumor tissue. The number of slides required for DNA extraction depends on how large the foci of normal and tumor tissue are see Note 3 . We recommend that at least one cm2 of normal and tumor tissue be used. 2. Stain the 5 im but not the 10 im slide with...
Harvesting the Xenograft Tumors
1. Xenografts may be harvested when an obvious subdermal growth is noticed. Typically at least 1 cm x 2 cm. Longer growth periods may result in larger tumors however, necrosis may be a concern. 2. A mouse with a xenograft growth is anesthetized as before. The growth area is sanitized with a 70 ethanol pad. The skin adjacent to the growth is pulled up with forceps and cut with scissors. Observation of the blood supply to the growth is made. This is referred to as the pedicle. The pedicle and a...
Isolation of Tumor DNA from Frozen Sections
1. Slides with 10 im thick frozen sections are immediately fixed in cold 100 ethanol for 15-30 s. 2. The section is then stained in 0.5 Toluidine blue for 8-10 s. Wash the slide twice in 100 ethanol, about 3 s per wash. Air dry slide at room temperature. 3. Using an H amp E stained reference slide, mark areas to be microdissected. Microdissect tissue using a scalpel and place in tube containing 500 L lysis buffer. 4. Digest overnight at 55 C in a shaking incubator. Store at 4 C until...
Materials 21 Reagents
2.1.1. Reagents for Tissue Culture 2. Penicillin-streptomycin, 100X Gibco-BRL . 3. L-glutamin, 100X Gibco-BRL . 4. Fetal bovine serum Gibco-BRL . 5. Phytohemagglutinin PHA Murex Biotech Ltd , Dartford, England, cat. no. HA15 . 6. Ethidium bromide, 10 mg mL, Gibco-BRL, cat. no. 5585UA . 7. Colcemid, 10 mg mL Boehringer Mannheim, cat. no. 295892 . 8. Methanol JT Baker, cat. no. 9093-03 . 9. Glacial acetic acid Malinckrodt, cat. no. UN2789 . 2.1.2. Reagents for Slide Processing 1. 20X SSC prepare...
Isolation and Purification of Intestinal Epithelial Cells Using Dispase and
1. Add 45 mg Dispase and 15 mg DNase to the combined epithelial cells final enzyme concentrations 3 and 1 mg mL, respectively and incubate in a 37 C water bath for 30 min. Vortex for 10 s at 5 min intervals. Use the minimum force required to vortex to minimize damage to cells. Small intestinal specimens almost invariably require longer stirring periods than large bowel specimens due to the release of many more epithelial cells from a comparable surface area as a result of the presence of villi....
for LCM RNA and Protein Isolation see Note 7
1. Ethanol-fixed frozen sections are dipped 15 times in RNase-free water using gloved hands or a slide holder. 2. 15 dips in hematoxylin stain. 3. The slide is dipped a few times in a deionized water bath to remove the majority of the stain, and is then dipped a few times in a fresh deionized water bath until the slide is clear of stain. 8. 15 dips in 95 ethanol, then repeat in a fresh 95 ethanol bath. 11. Air dry for at least 2 min or until the xylene is completely evaporated.