[Frontiers in Bioscience 2, a18-25, July 15, 1997]
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DISTINCT TUMOR SPECIFIC EXPRESSION OF TGFB4 (ebaf)*, A NOVEL HUMAN GENE OF THE TGF-b SUPERFAMILY

Siamak Tabibzadeh, Ravi Kothapalli , Ibrahim Buyuksal

Dept of Pathology, H. Lee Moffitt Cancer Center at the University of South Florida, College of Medicine, 12902 Magnolia Drive, Tampa, FL 33612

Received 7/10/97 Accepted 7/15/97 *: Patent pending

3. MATERIALS AND METHODS

3.1 Materials
A 1.1 kb cDNA fragment of GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was obtained from Clontech (Palo Alto, CA). Deoxycytidine 5 tri-phosphate dCTP a-32P (3000 Ci/mmol) was from Dupont NEN Research Products (Boston, MA). Prime-a-Gene labeling kit was from Promega (Madison, WI). RNA STAT-60TM was from Tell-Test, Inc (Friendwood, TX). Silane-coated, RNAse free, slides coated (Silane-PrepTM) for in situ hybridization and the Kodak-OMAT films were obtained from Sigma Chemical Company (St Louis, MO). Nick columns were obtained from Pharmacia Biotech (Piscataway, NJ). Digoxygenin labeling kit (SP6/T7) and DIG nucleic acid detection kit were from Boehringer Mannheim Corporation (Indianapolis, IN). All other chemicals were from either Sigma Chemical Company or Fisher Scientific (Pittsburgh, PA).

3.2 Processing of tissues
Tissues were obtained from the tumor bank at the H. Lee Moffitt Cancer Center according to the rules and regulations of the institution. Tumor tissues and, when available, normal tissues surrounding the tumor were frozen in liquid nitrogen and maintained at -70OC until used. Samples of the normal tissues and tumors were embedded in paraffin, sectioned and stained by hematoxylin and eosin for light microscopic examination and for establishing the diagnosis and determination of the tumor type.

3.3 Isolation of RNA and Northern blotting
RNA was isolated by acid guanidinium thiocyanate-phenol-choloroform extraction method as described (17). Briefly, the tissues were homogenized in RNA STAT-60TM. Each, 50-100 mg of tissue was homogenized in 1 ml of RNA STAT-60TM in a glass or Teflon Dounce homogenizer. Each homogenate was stored for 5 min at room temperature to permit the complete dissociation of nucleoprotein complexes. Then, 0.2 ml of chloroform was added for each ml of RNA STAT-60TMused. Each sample was covered and shaken vigorously for 15 seconds and allowed to stand at room temperature for 2-3 min. Following centrifugation at 12,000xg for 15 min at 4oC, each homogenate was separated into a lower phenol/chloroform phase and an upper aqueous phase. RNA in the upper aqueous phase was transferred to fresh tubes and mixed with isopropoanol to precipitate the total RNA. After centrifugation and drying, the precipitated RNA was dissolved in diethylpyrocarbonate (DEPC)-treated water by vigorous pipetting and by a gentle heating at 55o-60oC. The amount of RNA in each sample was determined spectrophotometrically. The quality of RNA was judged by the integrity of ribosomal. Northern blotting was done as described (18). Briefly, 20 mg of total RNA of each sample was denatured at 65oC in a RNA loading buffer, electrophoresed in 1% agarose containing 2.2 M formaldehyde gel, and blotted onto a Hybond nylon membrane using a positive pressure transfer apparatus (Posiblot, Stratagene, La Jolla, CA). The RNA was fixed to the membrane by UV crosslinking. Using the Prime-a-Gene kit, cDNA was labeled with [32P] to a high specific activity, and purified by Nick columns. Membranes were prehybridized in 50% formamide, 10x Denhardts solution, 4% saline sodium citrate (SSC), 0.05 M sodium pyrophosphate and 0.1 mg/ml of denatured Hering sperm DNA at 42oC for 2-4 hr and hybridized for 16 hr at 42oC with 106 cpm/ml of heat-denatured probe in the same buffer containing 10% dextran sulphate. Then, membranes were sequentially washed three times in 4x SSC, one time in 0.5x SSC and then one time in 0.1x SSC. All washes contained 0.1% sodium dodecyl sulphate (SDS), and were done at 65oC for 20 min each. The membranes were subjected to autoradiography at -70oC with intensifying screens. The same blot was stripped and reprobed for GAPDH. To reprobe a blot, the probe was stripped from the membrane in 75% formamide, 0.1x saline sodium phosphate ETDA (SSPE), and 0.2% SDS at 50oC for one hour.

3.4 In situ hybridization
Digoxigenin-labeled sense and anti-sense RNAs of TGFB4 (ebaf) were synthesized by in vitro transcription of the, full length cDNA, cloned into pBluescriptR SK- using digoxygenin dUTP. After alkaline hydrolysis, the probes were subjected to agarose gel electrophoresis to determine the size of the digested RNA fragments. Dot blotting was performed on the RNA fragments to insure that they were labeled. In situ hybridization was performed as previously described (19-20) Briefly, frozen sections of endometria were mounted on silane-coated, RNAse-free, slides and fixed in 4% formalin in PBS for 15 min at 4C. The tissue sections were rinsed in 2xSSC and then treated with proteinase K (1 mg/ml in 0.1 M Tris, 50 mM EDTA, 20 min, 37C) and acetylated for 10 min in 0.1 triethanolamine (pH 8.0), 0.9% sodium chloride and 0.25% acetic anhydride. The slides were dipped once in 2x SSC and then were dehydrated in ascending series of ethyl alcohol and air dried. The slides were prehybridized for 1 hr at 37C in 50% formamide, 1x Denhardts solution and 500 mg/ml tRNA, 0.3 M sodium chloride, 10 mM Tris, 1 mM EDTA (pH 8), and 10% dextran sulfate. Then, sections were incubated at 55C overnight in the same solution containing the appropriate concentration of the probe. The amounts of labeled probes needed were empirically determined first by a series of in situ hybridization experiments using various dilutions of the probes. Sense probe was used as the control. After hybridization, slides were washed three times for 10 min each at room temperature in 2x SSC, and the excess SSC was removed. The sections were then incubated with RNAse A (20 mg/ml) in 500 mM NaCl, 1 mM EDTA and 10 mM Tris HCl pH 8 at 37C for 30 min to remove the non-hybridized RNA. The sections were washed three times at room temperature, for 15 min each, in 2x SSC, 1xSSC, and 0.5x SSC and a final wash in 0.1x SSC at 55C for 45 min. Slides were washed in 100 mM Tris (pH 8), 150 mM sodium chloride for 10 min. Then, sections were blocked in 5% normal horse serum in the same buffer for 20 min at 37C. Slides were incubated with alkaline phosphatase labeled, anti-digoxygenin antibody for 1 hr at 37C, washed and developed in a mixture of Nitroblue tetrazolium salt (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP).