Dot Blot Hybridization Technique Research Articles

Differential regulation in the expression of hepatic genes in nephrotic and pair-fed rats.

The messenger ribonucleic acid (mRNA) levels of albumin, fibrinogen, alpha 1-acid glycoprotein (pAGP) and transferrin were analyzed in puromycin aminonucleoside (PAN)-treated (nephrotic) and in pair-fed (PF) rats with the Northern and dot blot hybridization techniques. Albumin mRNA levels in nephrotic and PF rats were 2- and 1.5-fold higher, respectively, than in ad-libitum-fed control (C) rats 6 days after PAN treatment. On day 11, this mRNA in PAN-treated rats was 2.5-fold higher than in PF and 4-fold higher than in C rats. A differential expression at the level of specific mRNAs was also detected for fibrinogen, pAGP, and transferrin in nephrotic and PF rats. On day 6, the fibrinogen and transferrin mRNA levels were significantly higher (p < 0.05) in nephrotic than in PF rats. In contrast, pAGP mRNA levels were normal or low in nephrotic rats and increased 2-fold in PF rats. These studies indicate the differential expression of hepatic genes in nephrotic and PF rats and show that albumin gene expression is only partially regulated by diet during the nephrotic stage of the disease.

Is human papillomavirus associated with cervical neoplasia in the elderly?

There have been no studies in the United States of human papillomavirus (HPV) in elderly women. This paper presents cross-sectional data on HPV and cervical neoplasia among 232 women age 65 or more. HPV deoxyribonucleic acid (DNA) testing was performed using a modified dot-blot hybridization technique. The prevalence of HPV DNA positivity was 3.5% (95% confidence interval (CI) 0.9%, 6.0%). There were six cases of histologic cervical neoplasia. The crude odds ratio for cervical neoplasia among HPV DNA positives was 18.3 (95% CI 2.8, 120.3). The adjusted odds, controlling for age, prior screening history, current sexual activity, and past contraception use, were 12.2 (95% CI 1.2, 122.9). Ever having had a Papanicolaou smear was protective, and there was a trend for the odds of having neoplasia to increase with age. Additional studies with larger samples of elderly women are needed. If confirmed, the results suggest that, independent of past screening, HPV may increase the risk of having cervical neoplasia for elderly women.

Quantitation of gene copy number and mRNA using the polymerase chain reaction.

Quantitation of the copy number of a specific gene in a given sample on the genomic as well as on the steady state RNA transcriptional level is of great interest in both basic and clinical research. Traditionally, the copy number of a gene in a given sample is estimated by Southern blot or dot blot hybridization techniques. A certain amount of total genomic DNA of a sample is digested with restriction enzymes, size fractionated by nondenaturing agarose gel electrophoresis, and transferred and permanently bound to a nitrocellulose or nylon membrane (Southern blot); alternatively, DNA can be transferred and bound to a membrane without previous manipulations (dot blot). The membrane is then incubated with a radioactive, labeled molecular probe, which specifically binds to the gene of interest. After washing steps to remove unbound probe, the membrane is exposed to film and a signal is detected. The intensity of the signal, which can be quantitated (e.g., by densitometry), correlates to the amount of specific...

Biological activity in the repopulating rat spermatocyte after the withdrawal of gossypol treatment: III. The activity for the transcription of messenger RNA

Mature male rats were treated with gossypol for 8 weeks after which the treatment was stopped to allow the arrested spermatogonia to revive. The arrested spermatogonia in the testes were allowed to develop for 15, 30, and 45 days. The amount of histone H 2b, H 4 and actin messenger RNA (mRNA) in the repopulated pachytene spermatocyte (RPS) and the control pachytene spermatocyte (CPS) was detected by the dot-blot hybridization technique. Histone H 2b mRNA in total testicular RNA is higher than that detected in CPS and RPS. The amount of histone H 2b mRNA in 5 μg to 10 μg of poly (A) + RNA isolated from CPS is 235% higher than that detected in the RPS. The H 2b mRNA content in the resting spermatocytes (15, 30, and 45 days after the withdrawal of gossypol treatment) was inhibited by 68, 72, and 45%, respectively. Histone H 2b mRNA transcription in the resting spermatocyte of these 3 time periods was inhibited by 26, 40, and 0%, respectively. Fifteen days after the withdrawal of gossypol treatment, the amount of actin mRNA in RPS was inhibited by 47%. The inhibitory effect of gossypol was rapidly recovered within 30 days after the withdrawal of the drug treatment. It was concluded that after the withdrawal of gossypol treatment, the production of H 2b, H 4, and actin mRNA in RPS was inhibited. However, the inhibitory effect on the mRNAs' production is reversible. The recovery rate for the mRNAs follows the following order: Actin mRNA > H 4 mRNA > H 2 b mRNA.

Detection of human papillomavirus DNA sequences in tongue squamous-cell carcinoma utilizing the polymerase chain reaction method.

Twenty-four cases of tongue squamous-cell carcinoma (SCC) were analyzed for human papillomavirus (HPV) DNAs by the polymerase chain reaction (PCR) method and the dot-blot hybridization technique. HPV DNAs were detected in 8 cases. One specimen histopathologically diagnosed as poorly differentiated grade-III SCC contained both HPV-16 and HPV-18 DNA, and 7 other cases contained HPV-16 DNA.

The decline of atrial natriuretic peptide (ANP) gene expression in older rats and the effects of ginsenoside on ANP gene expression

1. 1. The levels of atrial natriuretic peptide(ANP) gene expression in rat atria at 2–3 and 14–18 months of age and the effects of ginsenosides on r-ANP gene expression by determining the concentration of ANP-mRNA were investigated. The male and female rats were abdominally (i.p.) injected with aqueous solution of ginsenosides prepared from ginseng stems and leaves (G-PSL) and ginseng roots (G-PR), 50 mg/kg body wt, once a day for 7 days. Atria total RNA was extracted by the cold phenol method. The ANP-mRNA contents were determined using the Northern blot and dot blot hybridization technique with α-32 ∗P-labelled r-prepro-ANP-cDNA probe. 2. 2. The ANP-mRNA contents of 14–18 month rats were remarkably less than that of 2–3 month rats. The levels of male and female rats' atria at 14–18 months were about 15 and 60%, the content of male and female rats at 2–3 months respectively. 3. 3. G-PSL and G-PR increased the ANP-mRNA content of male rats at 14–18 months 1- and 2-fold, respectively, whereas G-PSL and G-PR decreased the ANP-mRNA content in male rats of age 2–3 months. 4. 4. These results revealed that the ANP gene expression declined during ontogenic ageing development and ginsenosides possessed anti-ageing effects in the heart endocrineous function aspect.

High prevalence of anti-hepatitis delta virus antibody in chronic liver disease in Somalia

We have assessed the prevalence of hepatitis delta virus (HDV) infection in people with histologically proven chronic liver disease living in Somalia. Among 104 patients studied (14 with chronic persistent hepatitis, 74 with chronic active hepatitis, and 16 with active cirrhosis), 52 were positive for hepatitis B surface antigen; of these, 26 (50%) carried anti-delta antibodies. HDV infection was detected more frequently in sera from hepatitis B e antigen (HBeAg) negative patients (60·9%) than in HBeAg positive patients (9·1%). Using the dot-blot hybridization technique, serum hepatitis B virus deoxyribonucleic acid was revealed in 73·1% of patients without HDV infection, while it was detected in only 7·7% of anti-delta positive patients. It is concluded that HDV is strongly associated with chronic liver disease in Somalia.

Molecular probe for identification of Trichomonas vaginalis DNA

Trichomoniasis is one of the most widespread sexually transmitted diseases in the world. Diagnosis can be achieved by several methods, such as direct microscopic observation of vaginal discharge, cell culture, and immunological techniques. A 2.3-kb Trichomonas vaginalis DNA fragment present in strains from diverse geographic areas was cloned and used as a probe to detect T. vaginalis DNA in vaginal discharge by a dot blot hybridization technique. This probe was specific for T. vaginalis DNA. It recognized strains from two regions in Italy (Sardinia, Piemonte) and from Mozambique (Africa). In addition, our probe did not cross-react with bacterial (Escherichia coli, Enterococcus spp., group B streptococci, Gardnerella vaginalis, Neisseria gonorrhoeae, Chlamydia trachomatis, and Lactobacillus spp.), viral (herpes simplex virus type 2), fungal (Candida albicans), protozoan (Entamoeba histolytica, Giardia lamblia, Plasmodium falciparum, Leishmania major, and Leishmania infantum), or human nucleic acids. The probe reacted with Pentatrichomonas hominis and Trichomonas foetus. The limit signal recognized by our probe corresponded to the DNA of 200 T. vaginalis isolates. The 2.3-kb probe was used in a clinical analysis of 98 samples. Of these, 20 samples were found to be positive both with the probe and by cell culture, and only 14 of these were positive by a standard wet mount method.

The effects of streptozotocin induced diabetes mellitus and fish oil compound on gene expression of atrial natriuretic peptide in rat

1. 1. Adult male Wistar rats were injected with streptozotocin (STZ: 55 mg/kg) for inducing diabetes. Then blood and atria for RNA extraction were withdrawn from rats treated 3 and 11 weeks previously with STZ respectively. Atrial total RNA were extracted with cold phenol method. The ANP mRNA contents were determined using Dot blot hybridization technique with alpha-32-P-labelled r-prepro ANP cDNA probe. 2. 2. Plasma glucose was increased and plasma immunoreactive insulin was lowered in rats at 3 and 11 weeks after injection of STZ. ANP gene expression in diabetic rats was depressed. ANP mRNA contents in rats treated 3 and 11 weeks with STZ were 86.4% and 31.7% of that of control rats. 3. 3. Three weeks after treatment of STZ, the rats were gastrically perfused with FOC (Fish Oil Compound) (0.355 ml/kg) once a day successively until 11 weeks. This treatment induces lower blood pressure in rats. ANP gene expression in FOC group was apparently recovered which had been decreased because of the effect of diabetes mellitus.

Androgen regulation of a specific gene in hamster flank organs

Flank organs of Golden Syrian hamster containing large sebaceous glands are a useful model for studying androgen-dependent gene expression. Recently, we have isolated an androgen-dependent gene from a cDNA library constructed from male mRNAs of flank organs. In the present study, using this gene as a probe, we examined the kinetics of loss and re-expression of the mRNA of this gene. Dot blot hybridization, the current method, can approximately quantify mRNA levels. Castration caused a marked decrease in the mRNA within a few days to undetectable levels. Topical application of testosterone re-activated the mRNA levels, with the earliest activation within 24 h. The results of various topical steroid applications showed a full re-activation of the mRNA by testosterone and dihydrotestosterone, partial re-activation by androstanedione and androstenedione, and no activation by androstanediol or dehydroepiandrosterone. Simultaneous application of testosterone and progesterone inhibited the expression of mRNA but application of dihydrotestosterone and progesterone did not. The current dot blot hybridization technique appears to be a more direct approach for studying androgen action on DNAs than other previously used methods such as morphometry or enzyme assay.

Carbon dioxide laser energy disperses human papillomavirus deoxyribonucleic acid onto treatment fields

The possibility of dispersing viral deoxyribonucleic acid during carbon dioxide laser treatment of human papillomavirus-containing genital infections has been investigated with a commercially available dot blot hybridization technique. The viral ribonucleic acid probes were specific for groups of human papillomavirus types 6/11, 16/18, and 31/33/35. Laser energy was delivered by continuous-wave mode and the plume of smoke was evacuated by a vacuum suction system. Samples were taken with Dacron swabs from lesional tissues of 43 patients as well as from the treated areas and from the 5 cm surrounding normal skin before and after laser vaporization. Human papillomavirus deoxyribonucleic acid was identified in swabs from 34 of 43 (79%) lesional tissues and 7 of 43 (16%) treatment fields. Although a trend for higher human papillomavirus deoxyribonucleic acid positivity in laser margins after therapy (7/43, 16%) than before (4/43, 9%) was observed, the rates were not statistically significant. It is concluded that carbon dioxide laser energy disperses human papillomavirus deoxyribonucleic acid onto treatment fields and the adjacent normal epithelium. Viral contamination of treated areas may be reduced by positioning the fume evacuator within 1 cm of the field of laser vaporization and cleaning the treated areas and surrounding tissue after therapy.

Two novel polyadenylation mutations leading to β+-thalassaemia

In an ongoing effort to identify point mutations causing beta-thalassaemia, we have found two previously unreported mutations which are located in the Poly A site of the beta-globin gene. The screening programme used amplified DNA and dot-blot hybridization with several 32P-labelled oligonucleotide probes. DNA samples which remained unidentified by this methodology were subjected to sequencing with 32P-labelled primers and modified T7 DNA polymerase. The newly discovered mutations were confirmed by the dot-blot hybridization technique. One type concerned an AATAAA----AATGAA mutation in the polyadenylation site and was found in one family from Yugoslavia (including one patient with the C----T mutation at codon 29 in trans), one from Bulgaria (the patient had the G----A mutation at IVS-I-110 in trans), and one from Greece (this patient had the C----G mutation at IVS-II-745 in trans). Haematological data for three simple heterozygotes suggested a rather mild beta(+)-thalassemia. The second type involved an AATAAA----AATAGA mutation and was found in one family from Malaysia. The propositus had the beta E mutation on the other chromosome, was originally diagnosed as mild Hb E-beta(+)-thalassaemia, and had Hb A and Hb E percentages which were nearly the same.

The effects of salt-loading and dehydration on atrial natriuretic peptide (ANP) gene expression

1. 1. Effects of sodium loading and dehydration on ANP gene expression were investigated in rats. 2. 2. ANP-mRNA was determined using Northern blot and dot blot hybridization technique with α- 32P-labeled r-preproANP-cDNA probe. Salt loading increased the ANP-mRNA content in atria. Correlation with ANP-mRNA content and the plasma sodium concentration was established. 3. 3. Deprivation of water for 2 and 4 days increased ANP-mRNA 2.1- and 1.6-fold, respectively. 4. 4. These results demonstrated that water-salt balance affects the ANP-gene expression.

SHORT COMMUNICATION: Absence of papillomavirus in skin tumors induced in SENCAR mice by a two-stage carcinogenesis protocol

SENCAR mice are unusually sensitive to induction of papillomas and squamous cell carcinomas by initiation with 7,12-dimethylbenzanthracene (DMBA) and promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA). Tumors induced by this protocol were tested for the presence of papillomavirus by immunohistochemistry, Southern blot, reverse Southern blot and dot blot hybridization techniques. Papillomavirus antigens were not detected in any of 235 tumors or 142 non-tumor-bearing skin samples analyzed. Southern blots and dot blots, using a mixed probe of cloned rodent papillomavirus DNA from the multimammate rat, Mastomys natalensis, and the European harvest mouse, Micromys minutus, did not reveal the presence of either episomal or integrated papillomavirus genomes in total cellular DNA extracts from the tumors or non-tumor-bearing skin. To circumvent the possibility that insufficient cross-homology existed to detect a papillomavirus genome with the mixed probe used, DNAs extracted from six papillomas were labeled and each used to probe reference blots that contained 25 cloned papillomavirus genomes excised from their vectors. No evidence for the presence of a papillomavirus genome was detected by this method. Therefore, it is unlikely that papillomaviruses play a role in the induction of tumors in SENCAR mice by two-stage carcinogenesis protocols.

Different pattern of expression of cellular oncogenes in human non-small-cell lung cancer cell lines

Altered and deregulated cellular oncogenes were found in many human solid tumors. Except for a few types of tumors that consistently exhibited specific altered proto-oncogenes, the majority of tumors are associated with a number of transcriptionally activated cellular oncogenes. In the heterologous group of non-small-cell lung cancer (NSCLC), nothing about a specific pattern of proto-oncogene expression is known. Therefore, we investigated the expression of a panel of cellular oncogenes in NSCLC cell lines. DNA and RNA from 11 established NSCLC cell lines (4 adenocarcinoma cell lines, 3 squamous cell carcinoma cell lines, 3 large-cell carcinoma cell lines and 1 mesothelioma cell line) were isolated and analysed using the Southern, dot blot and Northern hybridization technique. c-myc RNA expression was found in all NSCLC cell line, L-myc expression only in 1 adenocarcinoma cell line, N-myc and c-myb expression in none of the 11 cell lines examined. No c-myc amplification could be detected in the DNAs. v-sis-related mRNA was observed in 5/11 cell lines without association to a specific NSCLC subtype. v-src-related mRNA, found in all tested cells, exhibited increased levels in 1 adenocarcinoma cell line (A-549) compared to the other cell lines. Binding sites for epidermal growth factor (EGF) had been described previously in NSCL, therefore we found erbB homologue transcripts coding for the EGF receptor in all NSCLC cell lines. Also, c-raf1-, N-ras-, Ki-ras-, and H-ras-related RNA expression was observed in all lines. We conclude that L-myc, N-myc, and c-myb expression does occur less frequently in NSCLC than in SCLC. Also amplification does not appear to be an important mechanism by which the c-myc proto-oncogene is activated in NSCLC. A specific pattern of oncogene expression could not be detected in NSCLC cells; each cell line examined showed its own pattern. However, transcriptional activation of a proto-oncogene like erbB, ras, raf, src, and c-myc, which are all involved in the progression pathway of EGF, may be a common feature of NSCLC.

Detection of tomato spotted wilt virus using monoclonal antibodies and riboprobes

The immunoreactivity of a panel of monoclonal antibodies raised to tomato spotted wilt virus (TSWV) was examined in enzyme-linked immunosorbent assays (ELISA) and dot immunobinding assays (DIBA) procedures. MAbs 6.12.15 and 2.9 were specific for the nucleocapsid protein of TSWV. The sensitivity of the two immunoassays was compared with that of a dot-blot hybridization technique using riboprobes (RNA transcripts) to TSWV M RNA. Using deproteinized plant extracts or purified virus preparations, as little as 1 pg RNA could be detected. Although an ELISA using MAb 6.12.15, a DIBA procedure using MAb 3.22.6 and the dot-blot hybridization, detected several TSWV isolates in different host species equally well, the ELISA was most precise and most suitable for routine diagnosis in the field.

Effects of eicosapentaenoic and docosahexaenoic acids on apoprotein B mRNA and secretion of very low density lipoprotein in HepG2 cells.

Oleic acid (18:1n-9, OA), docosahexaenoic acid (22:6n-3, DHA), or eicosapentaenoic acid (20:5n-3, EPA) was added to HepG2 cells at a concentration of 1 mM in a 5:1 or 2:1 molar complex with bovine serum albumin (BSA), and this was incubated for 3 hours. The incorporation of 3H-glycerol into cellular and medium triglyceride (TG), and the mass of TG were measured. The effects of these fatty acids on the secretion of very low density lipoprotein (VLDL) apolipoprotein B (apo B) were estimated from the incorporation of 3H-leucine into the medium apo B in comparison to cells incubated with fatty acid-poor albumin. The secretion of human albumin by the cells was also estimated by immunochemical precipitation of the labeled albumin. In addition, the intracellular levels of apo B messenger ribonucleic acid (mRNA) were measured by the dot-blot hybridization technique. Relative to control cells incubated with BSA, OA (complexed to BSA at a 5:1 molar ratio) stimulated TG synthesis and secretion sevenfold. Compared to OA, EPA was 24% less effective for both processes, whereas DHA inhibited only the secretion of TG (-43%). The secretion of VLDL apo B was not affected by OA, but was decreased 31% by EPA and 54% by DHA. When the molar ratio of fatty acid complexed to albumin was changed to 2:1, similar results were obtained with respect to TG production. The levels of apo B mRNA relative to actin mRNA were not significantly altered by any of the fatty acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulatory effect of recombinant interleukin (il) 3 and il 4 on cytokine gene expression of bone marrow and peripheral blood mononuclear cells

This study shows that both recombinant human interleukin (rhIL)3 and rhIL4 induced proliferation in bone marrow (BM) cells of myelogenous leukemia patients in a manner similar to that reported using normal BM cells. However, we additionally found that these cytokines also influenced expression of other cytokines. Namely, using a reproducible dot blot hybridization technique we observed on the one hand that BM cells were capable of constitutively expressing low levels of cytokine mRNA coding for IL3, IL4, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte (G)-CSF and IL 1 beta, and on the other hand that in normal peripheral blood mononuclear cells rhIL4 inhibited mRNA expression coding for GM-CSF, G-CSF, IL3 and IL 1 beta, while IL4 mRNA and 28S rRNA was not affected. In contrast, rhIL3 marginally enhanced mRNA coding for IL3, GM-CSF, G-CSF and IL 1 beta and counteracted the inhibitory effect of IL4. In long-term cultures rhIL3 and rhIL4 had no significant effect on spontaneous cytokine gene expression of myelogenous leukemia-derived peripheral blood or BM cells, but made these cells more sensitive for subsequent stimulation with different polyclonal stimuli. Thus, IL3 and IL4 already modulate cytokine gene expression during the initiation of cell culture and differentiate BM cells into populations of cells which are capable of responding with an enhanced cytokine gene expression after polyclonal stimulation.

Detection of foot-and-mouth disease virus with DNA probes in bovine esophageal-pharyngeal fluids.

Infectivity and dot-blot hybridization techniques were compared for the detection of FMDV in esophageal-pharyngeal fluids from experimentally infected cows. The probe used includes the viral polymerase sequence which allows the detection of the three types of virus (A, O, and C) with equivalent sensitivity. Virus was detected by dot-blot hybridization as well as by infectivity, according to sample analysis of esophageal-pharyngeal fluids extracted seven days post-infection. It was not possible to recover infective virus from some samples extracted at 180 and 560 days post-infection, although specific viral RNA was detected by dot-blot hybridization. This could indicate the presence of a high ratio of non-infective viral mutants in FMDV carrier cattle. These results emphasize the usefulness of molecular hybridization techniques for FMDV carrier-state detection.

Detection of hepatitis B virus DNA in human serum by dot hybridization using a biotin-labelled probe.

A dot blot hybridization technique utilizing a biotin-labelled recombinant DNA probe was used to examine hepatitis B virus (HBV) DNA in serum. The lowest amount of HBV DNA in serum detectable by the color development of an avidin-biotin alkaline phosphatase complex was 40 picogram per 50 microliter. Validity of this method was confirmed by autoradiography using 32P-labelled and 3H-labelled HBV DNA probes. HBV DNA was found in 100% (34/34) of the HBsAg-positive and in 73.5% (25/34) of the HBsAg-negative subjects. In contrast, all nine cases showing negativity in HBV DNA were also HBsAg-negative. Correlation of HBe antigen/antibody with HBV DNA was investigated in 19 sera of which HBsAg was negative but anti-HBc-positive. Of 13 sera with anti-HBe 10 (76.9%) cases revealed HBV DNA positivity, while four (66.7%) of six sera without anti-HBe were positive in HBV DNA.In conclusion, serum dot hybridization assay utilizing a biotinylated probe proved useful in the detection of a free form of HBV DNA regardless of the presence of HBsAg and irrespective of HBeAg/anti-HBe status. Moreover, it is emphasized that practical advantages in speed, reproducibility, and safety have made this alternative even more attractive than autoradiography using radioisotope-labelled probes.