[Detection of HPV type 16 early genes E7 and E5 messenger RNA in uterine cervical neoplasia by reverse transcriptase polymerase chain reaction].
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Base Sequence↗
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Results for “Reverse Transcriptase Polymerase Chain Reaction”
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Detection of Norwalk virus in stool specimens by reverse transcriptase-polymerase chain reaction and nonradioactive oligoprobes.
A reverse transcriptase (RT)-polymerase chain reaction (PCR)-oligoprobe (OP), or RT-PCR-OP, method was developed for the detection of the Norwalk virus, which causes acute, epidemic gastroenteritis, in stool specimens. The Norwalk virus genome regions encoding the following two proteins were amplified by RT-PCR: the RNA polymerase (260-bp product) and a putative immunogenic protein (224-bp product). The resulting DNA fragments (amplicons) were hybridized to a digoxigenin-labeled internal OP specific to each amplicon. The detection limit of Norwalk virus, as determined by the endpoint of RT-PCR amplification for serially diluted, positive stool specimens, was similar to the actual virion titer as estimated by electron microscopy and at least 100-fold greater than the titer determined by radioimmunoassay (RIA). The RT-PCR-OP assay was specific for Norwalk virus and negative for other enteric viruses, including human and animal caliciviruses, hepatitis E virus, Snow Mountain agent, astroviruses, 16 human enteroviruses, and 5 human rotaviruses. Components of fecal specimens that interfere with RT-PCR were removed successfully by Sephadex G-200 gel chromatography. Of 20 stool specimens from human volunteers that were positive for Norwalk virus by RIA, a specific RT-PCR-OP result was obtained in 95% (19 of 20) of the samples by using the immunogenic protein primers and 75% (15 of 20) by using the polymerase primers. Twenty-six stool specimens from asymptomatic children and adults were negative by the Norwalk virus RT-PCR-OP. RT-PCR-OP detected Norwalk virus in the 4 of 21 coded fecal specimens that were also positive by enzyme immunoassay. Two samples that were positive by RIA or enzyme immunoassay were negative by RT-PCR, perhaps because viral RNA was not present or RT-PCR inhibitors were not adequately removed.
Adult↗
Quantitation of tropoelastin mRNA and assessment of alternative splicing in human skin fibroblasts by reverse transcriptase-polymerase chain reaction.
We have developed a reverse transcriptase-polymerase chain reaction (RT-PCR) assay for the quantitative measurement of levels of tropoelastin mRNA in total RNA preparations from skin fibroblasts. This method facilitates the reproducible detection of low abundance tropoelastin mRNA in the range of 10-1000 copies per cell. The procedure is based on a competitive RT-PCR assay where a tropoelastin cDNA-derived internal RNA standard is cotranscribed and coamplified together with the sample derived-endogenous target mRNA. In addition, RT-PCR of several domains of tropoelastin mRNA, followed by DNA sequence analysis of asymmetric PCR products, revealed a previously unknown pattern of alternate exon usage at the 3' end of the tropoelastin gene in human skin fibroblasts.
Alternative Splicing↗
Removal of inhibitory substances from human fecal specimens for detection of group A rotaviruses by reverse transcriptase and polymerase chain reactions.
A method was developed for the purification of rotavirus RNA from fecal extracts in order to permit the sensitive identification of group A rotavirus in fecal specimens by the polymerase chain reaction. Sequential reactions with reverse transcriptase and Taq polymerase with directed primers from rotavirus gene 6 yielded characteristic 259-base-pair fragments that were then visualized by silver stain on a polyacrylamide gel. As few as 500 genomic copies of purified rotavirus RNA could be detected in this manner. However, when the method was applied to fecal samples with added rotavirus virions, inhibition was noted in many of the fecal extracts which were tested. The inhibition could be reversed by dilution of the fecal extract, but sensitivity was also reduced by a corresponding dilutional factor. The inhibition was quantitatively removed by an added step in the extraction process that utilized chromatographic cellulose fiber powder (CF11 powder) to purify the rotavirus RNA during a series of rapid washing and elution steps. After CF11 purification, rotavirus RNA could be detected in experimental fecal samples at dilutions 1,000- to 10,000-fold beyond the detection limits of standard techniques such as enzyme immunoassay and the direct visualization of RNA following polyacrylamide gel electrophoresis. Furthermore, following purification by CF11, rotavirus RNA could be detected in all of seven enzyme-linked immunosorbent assay-positive fecal samples obtained from a child with rotavirus gastroenteritis; when CF11 purification was not performed, rotavirus RNA could be detected in only four of these samples, even after the removal of inhibitors by dilution of the extracts. Large-scale identification of rotavirus in fecal specimens may be possible by use of CF11 purification of viral RNA prior to sequential reactions with reverse transcriptase and Taq polymerase in a modified polymerase chain reaction.
Feces↗
Mechanism of androgen-induced thymolysis in rats.
To investigate the mechanism of androgen-induced thymolysis, the effects of various androgens, including testosterone (T), 19-nortestosterone, and 7 alpha-methyl-19-nortestosterone (MENT), were compared with those of estradiol and dexamethasone (DEX) in intact, castrated, and adrenalectomized male rats. The potency comparisons on thymus regression, based on mass of steroids, showed DEX to be the most potent, followed by estradiol and the androgens. Among the androgens, MENT was the most potent, followed by nortestosterone and T, an order similar to their anabolic potency on muscle. As the thymolytic effects of T and MENT were not altered by the concomitant administration of an aromatase inhibitor or a 5-reductase inhibitor, it was concluded that the effects of androgens were not mediated by their conversion to estrogens or 5 alpha-reduced metabolites. Involvement of glucocorticoid receptors in androgen action was excluded because mifepristone (an antiglucocorticoid) blocked DEX-induced, but not T- or MENT-induced, thymus regression. Flutamide, an antiandrogen, significantly blocked the thymolytic effect of T and MENT, providing further support for this conclusion. This suggested that the thymolytic action of androgens is an intrinsic property mediated via androgen receptors (AR). The occurrence of AR in the thymus was demonstrated by binding assays and the presence of AR messenger RNA (mRNA) by reverse transcriptase-polymerase chain reaction. Quantitative reverse transcriptase-polymerase chain reaction for AR mRNA in the thymus showed 6-fold more AR mRNA in the thymic epithelial cells than in the thymocytes. However, epithelial cells represent only a small fraction of the thymus. Hence, it is hypothesized that the androgens produce their thymolytic effects by stimulating the secretion of a factor(s) by the thymic epithelial cells that, in turn, causes regression of the thymus.
Adrenalectomy↗
Virologic features of an astrovirus diarrhea outbreak in a day care center revealed by reverse transcriptase-polymerase chain reaction.
Astroviruses cause outbreaks of diarrhea in children attending day care centers (DCCs). Reverse transcriptase-polymerase chain reaction (RT-PCR) was compared with EIA detection of astrovirus in stool specimens to characterize further the molecular epidemiology of an outbreak of astrovirus-associated gastroenteritis. Three hundred sixty-eight stool specimens collected prospectively from 36 children enrolled in a DCC during an 11-week outbreak of diarrhea were evaluated by EIA and RT-PCR. Astrovirus was detected in 32% of specimens by RT-PCR versus 10% by EIA (P < .001) and in 89% of children by RT-PCR versus 50% by EIA. The median duration of astrovirus excretion episodes detected by EIA was 1.5 days versus 4 days by RT-PCR (P = .06). Astrovirus was excreted for prolonged periods by immunocompetent children during this outbreak. RT-PCR was more sensitive than EIA for detection of astrovirus in stool specimens and redefined the epidemiology of astrovirus infection in this setting.
Age Factors↗
Tyrosine kinase inhibitor AG18 arrests follicle-stimulating hormone-induced granulosa cell differentiation: use of reverse transcriptase-polymerase chain reaction assay for multiple messenger ribonucleic acids.
A sensitive assay of multiple mRNAs by reverse transcriptase-polymerase chain reaction was adopted to study the hormonally regulated expression of steroidogenic enzymes in primary rat granulosa cells in culture. As little as 15-60 ng total RNA prepared from cultured cells were reverse transcribed in the presence of pd(T)6, and polymerase chain reaction was conducted in the presence of specific oligonucleotide pairs designed to identify cDNAs of steroidogenic enzymes. In combination with Northern blot analysis of cholesterol side-chain cleavage cytochrome P450 (P450scc) message, it is shown that a novel protein kinase inhibitor, tyrphostin AG18, arrests the FSH-induced accumulation of P450scc mRNA. This inhibition is dose dependent (IC50, 15 microM) and reversible. The addition of 80 microM AG18 to cells containing high levels of P450scc mRNA caused a rapid decline of the cytochrome message (t 1/2, 5 h), similar to the effect of 30 micrograms/ml alpha-amanitin. However, concomitant addition of the two drugs did not accelerate the mRNA degradation process, suggesting that AG18 does not affect message stabilization. Tyrphostin AG18 did not affect mRNA species that are not FSH inducible, such as the ribosomal protein L19, or the constitutively expressed low levels of steroid 5 alpha-reductase mRNA. Moreover, even the extremely high levels of P450scc mRNA in granulosa-lutein cells, being cAMP independent and terminally differentiated a few hours after LH surge, were not affected by the addition of AG18 in culture. In contrast, two additional key and FSH-inducible steroidogenic enzymes, i.e. aromatase cytochrome P450 and 3 beta-hydroxysteroid dehydrogenase-I, were inhibited by AG18 at their mRNA levels. These results suggest that an as yet undetermined tyrosine kinase pathway is involved in the cAMP-dependent signal transduction pathway of FSH action, so that the presence of AG18 does not allow FSH induction of gene expression to occur.
3-Hydroxysteroid Dehydrogenases↗
Detection of micrometastatic prostate cancer cells in lymph nodes by reverse transcriptase-polymerase chain reaction.
We have developed a highly sensitive method for detecting prostate cancer cells using reverse transcriptase-polymerase chain reaction (RT-PCR) with primers specific for prostate-specific antigen gene. Forty-four lymph nodes obtained from 22 patients with prostate cancers were analyzed by RT-PCR to detect metastatic prostate cancer cells. RT-PCR could detect prostate-specific antigen mRNA in five lymph nodes with histologically and/or immunohistochemically identifiable metastases and in four lymph nodes with negative histological and immunohistochemical analyses for metastases. RT-PCR was a more sensitive method than histology and immunohistochemistry in detecting metastatic prostate cancer cells and could be applied for diagnosing micrometastases of prostate cancer to lymph nodes. This highly sensitive RT-PCR will be a relevant tool to allow a more accurate clinical assessment of lymph node metastases of prostate cancer and to understand lymphatic dissemination of prostate cancer biologically.
Base Sequence↗
Detection of breast cancer micrometastases in axillary lymph nodes by means of reverse transcriptase-polymerase chain reaction. Comparison between MUC1 mRNA and keratin 19 mRNA amplification.
Usefulness of MUC1 mRNA and keratin 19 mRNA as a target of reverse-transcriptase polymerase chain reaction (RT-PCR) was compared in the detection of breast cancer micrometastases in axillary lymph nodes. RT-PCR amplification of MUC1 mRNA and keratin 19 mRNA was conducted using total RNA samples. RT-PCR products were stained with ethidium bromide and analyzed by agarose gel electrophoresis. Expression of both MUC1 mRNA and keratin 19 mRNA was detected by RT-PCR in a breast cancer cell line (MRK) and in all the 23 primary breast cancers but not in the control lymph nodes obtained from patients with benign diseases. A serial dilution study of MRK cells against normal lymph node cells has shown that detection sensitivity of MUC1 RT-PCR and keratin 19 RT-PCR were 1/10(5) and 1/10(6) (cancer/lymph node cells), respectively. Sixty-three axillary lymph nodes were obtained from 23 patients with primary breast cancer, and metastases in each lymph node were investigated by histological examination (hematoxylin and eosin sections) and RT-PCR method. In all 10 lymph nodes, which were histologically metastasis-positive, both MUC1 mRNA and keratin mRNA were detected by RT-PCR. Of the 53 histologically negative lymph nodes, 3 (6%) and 5 (9%) lymph nodes were found to express MUC1 mRNA and keratin 19 mRNA, respectively, indicating the presence of micrometastases which could be detected by RT-PCR but not by histological examination. These results demonstrate the usefulness of both MUC1 RT-PCR and keratin 19 RT-PCR in the detection of breast cancer micrometastases in lymph nodes, and also indicate the superiority of keratin 19 RT-PCR over MUC1 RT-PCR because of its higher detection sensitivity.
Axilla↗
Quantitative reverse transcriptase-polymerase chain reaction of glucose transporter 1 mRNA levels in rat brain microvessels.
We investigated the usefulness of reverse transcriptase-polymerase chain reaction (RT-PCR) to quantify glucose transporter 1 (GLUT1) mRNA in cerebral microvessels. The technique was validated using an in vitro-transcribed RNA fragment (riboprobe) of partial 3' noncoding sequence of rat brain GLUT1 gene. A known amount of the riboprobe was reverse-transcribed to cDNA (target DNA). PCR primers were made to amplify a 292-bp fragment of the target DNA. The 5' primer was end labeled with 32P. An oligonucleotide of 100 bp containing the same sequences as the first 30 and the last 70 bases of the 292-bp fragment of the target DNA was synthesized and used as competitive DNA. The target DNA was coamplified with increasing amounts of competitive DNA using the same two primers. The ratio of radioactivity between amplified products of the target DNA (292-bp fragment) and the competitive DNA (100-bp fragment) was determined quantitatively after separation by gel electrophoresis and radioactivity counting. This method gave an accurate estimation of the amount of the riboprobe in the reaction and a 2- to 5-fold change in the amounts could be detected. By this method, the mean amount of GLUT1 mRNA from purified rat brain microvessels was estimated to be 1.5 +/- 0.1 x 10(-6) ng/ng total RNA. This value was about 10-fold higher than that in rat cell line PC12.
Animals↗
Localization of 12-lipoxygenase mRNA in cultured oligodendrocytes and astrocytes by in situ reverse transcriptase and polymerase chain reaction.
We studied the distribution of 12-lipoxygenase mRNA in glial cells. First, mRNA was detected from cellular extracts by soluble-phase reverse transcriptase-polymerase chain reaction (RT-PCR). Taking into account that cell culture populations could not be 100% homogeneous, we then developed, for the first time, an in situ RT-PCR combined with immunocytochemistry with cell specific markers. Using this procedure we showed that 12-lipoxygenase mRNA was expressed both in mature oligodendrocytes and astrocytes.
Animals↗
Reverse transcriptase-polymerase chain reaction detects induction of cardiac-like alpha myosin heavy chain mRNA in low frequency stimulated rabbit fast-twitch muscle.
Using reverse transcriptase-polymerase chain reaction we quantified in rabbit skeletal muscles expression levels of the highly homologous cardiac alpha and beta myosin heavy chain (alpha MHC, beta MHC) mRNA isoforms. Masseter muscle displayed highest levels of a cardiac-like alpha MHC mRNA. This isoform was present at 20-fold lower amounts in slow soleus and at 200-fold lower levels in several fast-twitch muscles. Low-frequency stimulation periods exceeding 20 days drastically induced the alpha MHC mRNA in fast tibialis anterior. The alpha MHC mRNA was 140-fold elevated after 60 days when beta MHC mRNA had increased 50-fold. Our results demonstrate the wide distribution of a cardiac-like alpha MHC mRNA in skeletal muscle and its marked induction during fast-to-slow transition as induced by low-frequency stimulation.
Animals↗
Comparative evaluation of bovine immunodeficiency-like virus infection by reverse transcriptase and polymerase chain reaction.
Infection of embryonic bovine lung (EBL) cells by bovine immunodeficiency-like virus (BIV) were monitored by reverse transcriptase (RT), syncytia formation and polymerase chain reaction (PCR). Infection can be detected by PCR at 24 h while the presence of syncytia and RT were not detected until much later. The detection of BIV RT can be optimized by changing the pH and salt conditions. The enzyme is very sensitive to changes in pH but can tolerate a wider range of salt and MgCl2 concentrations. Infection of primary human cell cultures by BIV was monitored by both PCR and RT. No active infection of human cells were detectable.
Animals↗
Detection of retinoic acid receptor mRNA in rat tissues by reverse transcriptase-polymerase chain reaction.
The presence of retinoic acid receptor (RAR) alpha, beta and gamma mRNA was examined in 16 different kinds of rat tissue using the highly sensitive reverse transcriptase-polymerase chain reaction technique. The data demonstrated that each tissue expressed at least two types of RAR mRNA. Among the three types of RAR mRNA, RAR alpha was widely expressed in all types of organ and was the dominant form expressed in the gastrointestinal tract. RAR beta mRNA was not present in the intestine and spleen. In addition, RAR beta mRNA levels were high in the heart, lung, brain, testis and epididymis. RAR gamma mRNA was abundant in both male and female reproductive systems, as well as epidermal tissues. The prevalence of each RAR mRNA in the tissues suggests the diverse biological roles of these receptors.
Animals↗
Detection of the EWS/WT1 gene fusion by reverse transcriptase-polymerase chain reaction in the diagnosis of intra-abdominal desmoplastic small round cell tumor.
We report two cases of intra-abdominal desmoplastic small round cell tumor with characteristic clinical, histological, immunohistochemical, and ultrastructural features. Fusion of the EWS gene on chromosome 22 and the WT1 gene on chromosome 11, resulting from the chromosomal translocation t(11;22)(p13;q12), was detected by reverse transcriptase polymerase chain reaction (RT-PCR) in both cases. This translocation has been previously reported in this type of tumor using either cytogenetic or molecular biological techniques. Tumor tissue from both cases revealed no chimeric fusion transcripts characteristic of the Ewing sarcoma family of peripheral primitive neuroectodermal tumors or of alveolar rhabdomyosarcoma, two tumors in the differential diagnosis of intra-abdominal desmoplastic small round cell tumor. This report demonstrates the utility of molecular studies as an adjunct in the diagnosis of this rare and aggressive tumor.
Abdominal Neoplasms↗
Ultra-rapid, simple, sensitive, and economical silica method for extraction of dengue viral RNA from clinical specimens and mosquitoes by reverse transcriptase-polymerase chain reaction.
A rapid, simple and efficient single-tube procedure is described for the isolation of dengue virus RNA from small amount of serum (10 microliters) followed by a reverse transcriptase-polymerase chain reaction (RT-PCR). Recovery of RNA is based on the lysing and nuclease-inactivating properties of guanidinium thiocyanate in the presence of silica. The silica RT-PCR can be completed within 5 hours starting from RNA extraction to agarose gel electrophoresis. All of the 63 dengue-3 culture-positive sera were RT-PCR-positive (virus titres: < 10(2) to 11(10.69.). Of 33 culture-negative acute sera from serologically confirmed dengue fever patients collected during dengue-3 epidemic, 4 were RT-PCR-positive. RT-PCR was also positive in 29 of 30 dengue-1 culture-positive sera (virus titres range: < 10(2) to 10(8.69). Dengue-1 virus was also detected in field-caught Aedes aegypti mosquitoes by silica RT-PCR.
Aedes↗
Detection of neuroblastoma cells in bone marrow and peripheral blood at diagnosis by the reverse transcriptase-polymerase chain reaction for tyrosine hydroxylase mRNA.
BACKGROUND: Bone marrow metastasis often occurs in patients with neuroblastoma; therefore, a sensitive assay to detect occult neuroblastoma cells in bone marrow (BM) and peripheral blood (PB) is needed. The feasibility and clinical value of using the reverse transcriptase-(RT) polymerase chain reaction (PCR) to amplify mRNA for tyrosine hydroxylase (TH), the first enzyme of catecholamine synthesis, was evaluated to detect neuroblastoma cells in patient samples. METHODS: Thirty-eight patients with Stages I to IV neuroblastoma and eight healthy donors were included in this study. Bone marrow and PB samples obtained at diagnosis were examined for TH mRNA. After preparation of complementary DNA, the PCR was performed to amplify the TH gene. RESULTS: Tyrosine hydroxylase mRNA was detected in neuroblastoma samples including a cell line and tumor tissues, but was not detected in normal BM or PB mononuclear cells. Neuroblastoma cells were detected at a level of 1 per 10(5-6) normal PB mononuclear cells by this method. Tyrosine hydroxylase mRNA was detected in 18 of 38 BM samples, and all 12 BM samples with cytologic evidence of tumor cells were positive for TH mRNA by the RT-PCR. Six of 26 patients without cytologic evidence of tumor cells in the BM were also positive for TH mRNA. TH mRNA was detected in BM samples from 1 of 14 patients with Stage I disease, 2 of 7 patients with Stage II disease, 1 of 3 patients with Stage III disease and all patients with Stage IV (11 patients) and IVS (3 patients) diseases. Tyrosine hydroxylase mRNA also was detected in 8 of 14 PB samples (one of five patients in Stages I, II or III, and 7 of 9 in Stage IV or IVS). CONCLUSIONS: Reverse transcriptase-polymerase chain reaction amplification of TH mRNA was a sensitive and specific method of detecting occult neuroblastoma cells in BM and PB samples. Neuroblastoma cells could be detected by this method in some BM samples that had no cytologic evidence of tumor cells and in some PB samples at the time of diagnosis. The clinical significance of these very low levels of neuroblastoma cells detected by RT-PCR requires further investigation.
Base Sequence↗
Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction.
We report on the development and application of a rapid assay for detecting and typing dengue viruses. Oligonucleotide consensus primers were designed to anneal to any of the four dengue virus types and amplify a 511-bp product in a reverse transcriptase-polymerase chain reaction (PCR). First, we produced a cDNA copy of a portion of the viral genome in a reverse transcriptase reaction in the presence of primer D2 and then carried out a standard PCR (35 cycles of heat denaturation, annealing, and primer extension) with the addition of primer D1. The resulting double-stranded DNA product of the RT-PCR was typed by two methods: dot blot hybridization of the 511-bp amplified product to dengue virus type-specific probes or a second round of PCR amplification (nested PCR) with type-specific primers, yielding DNA products the unique sizes of which were diagnostic for each dengue virus serotype. The accumulated data demonstrated that dengue viruses can be accurately detected and typed from viremic human serum samples.
Animals↗