Quantitative reverse-transcriptase polymerase chain reaction assay for mRNA levels of steroid 5alpha-reductase isozymes.
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3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
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Results for “Reverse Transcriptase Polymerase Chain Reaction”
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Use of a reverse-transcriptase polymerase chain reaction for monitoring the shedding of feline coronavirus by healthy cats.
The pattern of shedding of feline coronavirus (FCoV) was established in 155 naturally infected pet cats from 29 households over periods of up to five years. Viral RNA was detected in faeces by reverse-transcriptase PCR (RT-PCR), and plasma antiviral antibodies by immunofluorescence. The cats rarely shed FCoV in their saliva. Three patterns of FCoV shedding were observed. Eighteen of the cats shed virus continuously, so were persistent, and possibly lifelong, carriers; none of them developed feline infectious peritonitis. Fifty-six cats ceased shedding virus, although they were susceptible to reinfection, and 44 shed intermittently or were being continuously reinfected. Four of the cats were resistant to infection. Seventy-three per cent of the virus shedding episodes lasted up to three months and 95 per cent up to nine months. There was a correlation between shedding and antibody titre but the cats could remain seropositive for some time after they had ceased shedding virus. One-off testing for FCoV by RT-PCR is inappropriate. Identification of longterm carriers requires that a positive result be obtained by RT-PCR on faecal samples for at least eight consecutive months. A cat should be shown to be negative over five months, or to have become seronegative, to ensure that it has ceased shedding virus.
Animals↗
Design of an antisense reverse-transcriptase-polymerase chain reaction primer efficient for all hepatitis C virus genotypes: comparison of its performance vs a commercial primer.
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Base Sequence↗
Rapid detection of African horsesickness virus by the reverse transcriptase polymerase chain reaction (RT-PCR) using the amplimer for segment 3 (VP3 gene).
The complete sequence of the major core protein (VP3) gene of African horsesickness virus serotype 4 (AHSV-4; vaccine strain) was determined by analysis of a complete cDNA clone representing segment 3. The RNA was 2,789 bp long and a comparison of its sequence with that of bluetongue virus serotype 10 (BTV-10) revealed 58% nucleotide similarity. Based on these data, the reverse transcriptase-polymerase chain reaction (RT-PCR) technique was applied to the specific detection of AHSV using a pair of primers designed for AHSV-4 VP3 gene. Approximately 230 bp of PCR products were amplified by RT-PCR from the total RNA extracts (mRNA and dsRNA) of Vero cells infected with eight serotypes of AHSV. No product was observed analogous to other orbiviruses. The supernatant of the infected cell culture fluid without any RNA purification was also suitable as a template for RT-PCR after being denatured at 94 degrees C for 5 min. The sensitivity of this method was between 10(0) and 10(1) TCID50 when viral RNA from the supernatant of infected cell culture was subjected to RT-PCR. The whole procedure for detecting the virus RNA by RT-PCR could be carried out within 5 h. The RT-PCR with AHSV VP3 gene as a target was found to be a simple, highly specific and sensitive assay for AHSV.
African Horse Sickness Virus↗
Genotyping of Plasmodium falciparum gametocytes by reverse transcriptase polymerase chain reaction.
A molecular assay has been developed for the specific detection and genetic characterisation of Plasmodium falciparum gametocytes in the blood of malaria infected individuals. The assay is based on the reverse transcription and polymerase chain reaction (RT-PCR) amplification of the messenger RNA of gene pfg377, a sexual-stage specific transcript abundantly produced in maturing gametocytes. The gene contains four regions of repetitive sequences, of which region 3 was shown to be the most polymorphic in laboratory clones and field isolates of the parasite. Analysis of samples of malaria infected blood by RT-PCR specific for region 3 has enabled identification of multiple gametocyte-producing clones within single infections. The assay is able to detect gametocytes below the threshold of microscopic detection, and is highly specific for its gametocyte targets also in the presence of a vast excess of asexual forms.
Alleles↗
Screening for cytokine mRNA in human villous and extravillous trophoblasts using the reverse-transcriptase polymerase chain reaction (RT-PCR).
During the process of placental implantation, sessile villous trophoblast cells migrate from the villi into the decidua as isolated motile extravillous trophoblast cells. There is differential expression of the epidermal growth factor-receptor (EGF-R) and c-erbB2 proteins on villous and extravillous trophoblast populations. Using monoclonal antibodies to EGF-R and c-erbB2, we have obtained highly purified populations of villous and extravillous trophoblast by fluorescence activated cell sorting. These cells were examined by the reverse transcriptase-polymerase chain reaction (RT-PCR) using nested internal primer pairs for the following cytokines: CSF-1, GM-CSF, TNF-alpha, TGF-beta1, IFN-gamma, IL-2, LIF and also for LIF-receptor. TNF-alpha and TGF-beta 1 were present in all trophoblast populations. GM-CSF and CSF-1 were only found in some samples, with preferential expression of CSF-1 in villous populations. IFN-gamma, IL-2 and LIF mRNA were not found, although all samples contained LIF-receptor mRNA. These cytokines (CSF-1, TGF-beta, TNF-alpha and GM-CSF) are likely to influence trophoblast growth and differentiation in an autocrine manner, since their receptors are also present on trophoblast. These results illustrate a quick and simple method to analyse for the presence of cytokine and other transcripts in trophoblast subpopulations during early pregnancy.
Base Sequence↗
mRNA expression, measured by quantitative reverse transcriptase polymerase chain reaction, of five putative drug resistance parameters, in normal and leukaemic peripheral blood and bone marrow.
Using a quantitative reverse transcriptase PCR assay, the mRNA expression of five putative drug resistance-related genes were assessed in normal peripheral (n = 14) and bone marrow (n = 4) mononuclear cells from healthy donors and patients with acute myeloid leukaemia (n = 11). The mRNA levels of MDR1, the multidrug resistance-associated protein and glutathione-S-transferase pi were equally expressed in both compartments. Bcl-2 mRNA was slightly higher in the leukaemic marrow samples. However, topoisomerase II alpha mRNA levels were found to be much higher in normal and leukaemic marrow cells compared to peripheral blood (p < 0.01), which may, in part, reflect the different proliferation pattern of the mononuclear cells in the two compartments. Such findings could be important for researchers using bulk assays in a mix of samples from peripheral blood or bone marrow to investigate prognostic factors in patients with leukaemia.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
[Use of in situ hybridization and reverse transcriptase polymerase chain reaction in situ methods for studying infection caused by the hepatitis C virus].
Low level of hepatitis C virus (HCV) replication in hepatocytes does not provide reliable morphological or ultrastructural criteria for detecting an HCV infection by optic and electron microscopy. On the other hand, detection of cellular and subcellular location and tropism of HCV and comparison of virus replication in infected tissues with the level of viremia in patients with hepatitis C require modern methods, combining morphological and molecular biological aspects of diagnosis, such as in situ hybridization (ISH) and reverse transcription polymerase chain reaction in situ (RT-PCR in situ). The target for ISH is HCV RNA, while for RT-PCR in situ it is amplified HCV cDNA, which notably improves the sensitivity of hybridization.
Hepatitis C↗
Detection of leukemic blasts in peripheral blood specimens by reverse transcriptase polymerase chain reaction assay for myeloperoxidase mRNA.
The enzyme myeloperoxidase (MPO), an important constituent of granulocytes, is used clinically in the diagnosis and classification of acute leukemia. Whereas MPO protein or enzyme activity is detectable in mature granulocytes, MPO RNA is present only in myeloblasts or promyelocytes. Some undifferentiated, biphenotypic, or lymphoblastic leukemias, although lacking MPO enzyme activity, express low levels of MPO RNA, a fact that may be of prognostic and therapeutic importance. However, current methods are inadequate for reliably detecting low levels of MPO RNA in leukemic cells containing few copies of the message. We have developed a new and highly sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) assay for detecting low levels of MPO RNA in peripheral blood specimens of leukemic patients. This report describes the assay, and demonstrates its potential applicability to the diagnosis and classification of acute leukemias.
Base Sequence↗
Quantification of ribonucleotide reductase expression in wild-type and hydroxyurea-resistant cell lines employing in situ reverse transcriptase polymerase chain reaction and a computerized image analysis system.
Ribonucleotide reductase (RR) is the enzyme responsible for converting nucleoside diphosphates to deoxynucleoside diphosphates, ensuring a balanced supply of deoxyribonucleotides for DNA synthesis. Expression of RR is tightly regulated, but it is affected by exogenous agents, such as hydroxyurea (HU), which inactivates the tyrosyl free radical on the small subunit of RR, R2. We have previously employed in situ reverse transcriptase (RT)-PCR to estimate expression of R2 in wild-type and HU-resistant human colon carcinoma cell lines and to correlate altered expression of R2 with changes in cell size and morphology. The current studies were undertaken to render this methodology more quantitative. Both wild-type and resistant cells were grown on partitioned glass slides and analyzed with in situ RT-PCR. Because both wild-type and resistant cells were analyzed under a single cover slip, protease digestion, reverse transcription, PCR, and color development were all performed under identical conditions. Images were analyzed with NIH Image 1.59 software. There was a highly significant correlation between expression of R2 and cell size for both sensitive and resistant cells (P = 0.0001, for both). When cell size was compared either with expression of R2 or cell shape, however, these correlated only in wild-type cells (P = 0.001 and 0.0001, respectively). These data demonstrate that normal cell growth in the unperturbed wild-type cell line was closely linked to expression of R2, whereas in the resistant variants which overexpress R2, these correlations were absent, suggesting that HU resistance is related to loss of linkage between R2 expression and cell growth and confirming previous data relating overexpression of R2 with multiple other changes in the cell growth repertoire. Thus, we have demonstrated for the first time a quantitative application of in situ RT-PCR.
Base Sequence↗
A novel, non-nested reverse-transcriptase polymerase chain reaction (RT-PCR) test for the detection of the t(15;17) translocation: a comparative study of RT-PCR cytogenetics, and fluorescence In situ hybridization.
BACKGROUND: The development of a rapid and simple reverse-transcription polymerase chain reaction (RT-PCR) assay is described that identifies the promyelocytic leukemia- retinoic acid receptor alpha (PML-RARa) hybrid messenger RNA (mRNA), a characteristic feature of acute promyelocytic leukemia (APL). METHODS AND RESULTS: Randomly primed complementary (cDNA) is synthesized from leukocyte RNA and amplified in the presence of Taq Gold in 2 separate reaction tubes containing primer pairs specific for intron 3 (bcr 3, long [L] form mRNA transcript) and intron 6 (bcr 1, short [S] form)/exon 6 (bcr 2, variant [V] form) breakpoints in PML, respectively. The different sized products generated from each RNA transcript (S, L, or V forms) are readily and unambiguously distinguishable after agarose gel electrophoresis without the need for either nested PCR or hybridization. The sensitivity of the assay is 1 in 10,000 to 1 in 100,000. The separate amplification of a b2-microglobulin transcript controls for adequate RNA and cDNA preparation. The newly developed assay was used clinically for the evaluation of 78 patients with APL. It was rapid and more sensitive than cytogenetic karyotyping, both for the diagnosis of APL and the assessment of minimal residual disease (MRD) after therapy. RT-PCR detected PML-RARa mRNA in all cases positive for the t(15;17) translocation by cytogenetics. However, as many as 50% and 80% of the diagnostic specimens and the specimens for MRD assessment, respectively, that were positive by RT-PCR were negative by cytogenetics. The ratio of cases with L-form to S-form PML-RARa fusion transcript was 2:1, whereas 3 cases (10%) had fusion sites in exon 6 of the PML gene (V forms). In addition, approximately 50% of the patients were diagnosed morphologically with microgranular M3V-type leukemia, but no significant correlation with PML breakpoints was found. CONCLUSION: The current assay is rapid, sensitive, and specific without using nested PCR or hybridization.
Biomarkers, Tumor↗
A real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) to detect breast carcinoma cells in peripheral blood.
BACKGROUND: The detection of occult carcinoma cells in patients with breast cancer has been shown to predict disease recurrence and metastasis. MATERIALS AND METHODS: To improve on molecular detection of breast carcinoma cells in blood, we have developed a sensitive and quantitative assay using real-time quantitative RT-PCR identifying transcripts of the cytokeratin-19 (CK19) gene. RESULTS: This real-time quantitative RT-PCR is sensitive, accurate and has a high reproducibility within a wide dynamic range, which permits simultaneous quantitative analysis of samples with varying input concentrations. Furthermore, the procedure offers several technical advantages over classic quantitative PCR methods (competitive RT-PCR, Northern blotting) such as decreased likelihood of contamination due to absence of post-PCR manipulations, high sample throughput because of absence of post-PCR processing time (no agarose gel electrophoresis). In this pilot study, we detected significantly elevated CK19 transcript levels in < 10% of the volunteers, in +/- 30% of stage I-IIIa patients preoperatively and in > 70% of the and stage IV breast cancer patients. CONCLUSIONS: Analyses using this real time quantitative RT-PCR for CK19 mRNA may prove to have clinical implications in the assessment of circulating tumour cells in peripheral blood, micrometastases in bone marrow or lymph nodes in breast cancer patients. Application of this technique in a clinical population may improve diagnosis and monitoring of metastatic breast cancer and its validation is currently ongoing.
Adult↗
Semi-quantitative reverse-transcriptase polymerase chain reaction: an approach for the measurement of target gene expression in human brain.
Polymerase chain reaction (PCR) is a very powerful tool for qualitative evaluation of nucleic acids due to its high efficiency and convenience. Together with the reverse transcription (RT) reaction, the PCR method has been widely applied to the quantitative measurement of DNA and RNA messages. Since RT-PCR is much more sensitive than all of the traditional methods for quantification of mRNA, including Northern blot, ribonuclease protection, RNA blot, and solution hybridization assays, it is the method of choice for quantitative analyses of low abundance mRNA messages. However, because of the exponential nature of the PCR amplification, RT-PCR quantitation may be problematic, giving false estimates of the abundance of the target messages. By using the constitutively expressed 'housekeeping' gene cyclophilin as a reference gene to normalize mRNA levels, and by taking data from the exponential phase of the PCR amplification, we have developed a rapid and reliable semi-quantitative measurement of the relative abundance of dopamine transporter (DAT) mRNA. The semi-quantitative PCR method has been applied to illustrate its use for the measurement of DAT mRNA in post mortem human brain.
Adult↗
Discriminative quantification of cytochrome P4502D6 and 2D7/8 pseudogene expression by TaqMan real-time reverse transcriptase polymerase chain reaction.
The human drug oxidizing cytochrome P450, CYP2D6, is expressed at highly variable levels mainly due to a common genetic polymorphism which leads to the poor metabolizer phenotype in carriers of two nonfunctional alleles and to the extensive metabolizer phenotype in carriers of one or more functional alleles. Investigation of the role of CYP2D6 mRNA for expression and the possibility of using mRNA expression as a surrogate marker has been hampered by the presence of two pseudogenes, CYP2D7P and CYP2D8P. We therefore developed highly specific TaqMan real-time reverse transcriptase-PCR assays for the discriminative quantification of CYP2D6 and CYP2D7/8P transcripts. By in vitro transcription of plasmids containing the CYP2D6 cDNA or a hybrid CYP2D6/7 cDNA constructed by in vitro mutagenesis, authentic cRNAs were synthesized to be used for specificity testing and for absolute quantification. The method was used to determine CYP2D transcripts in a large number of human livers samples. CYP2D6 was not normally distributed with a median mRNA content of 3.2 transcripts per picogram of total RNA in all livers (range 0.32-14.8, N = 74). Expression in genetic poor metabolizers (1.81, N = 6) was significantly lower compared to extensive metabolizers (3.33, N = 68, P = 0.022). Similar expression levels were found for CYP2D7/8P (median 3.38 transcripts/pg, range 0.46-14.3), which were correlated to CYP2D6 mRNA (r(S) = 0.46, P < 0.0001) but did not depend on CYP2D6 genotype. These data demonstrate genotype-dependent mRNA expression for CYP2D6 and they emphasize the necessity of differentiating between the functional CYP2D6 and the CYP2D pseudogenes.
Base Sequence↗
[Diagnosis of Prader-Willi syndrome by reverse transcriptase polymerase chain reaction].
To approve Prader-Willi syndrome by molecular diagnostic assay, polymerase chain reaction of reverse-transcribed RNA was introduced by which indirect information can be gained on all known forms of the mutation. In this pilot study, 4 patients and 16 healthy control individuals were examined. Although the different mutation forms can not directly by identified by this approach, it is a useful and reliable test to confirm the clinical diagnosis of the Prader-Willi syndrome, and to screen for the syndrome in patients who present with only a few typical features.
Humans↗
Detection of molecular variants of BCR-ABL gene in bone marrow and blood of patients with chronic myeloid leukemia by reverse-transcriptase polymerase chain reaction (RT-PCR).
Very limited data exists in Thailand regarding the frequency of BCR-ABL leukemic gene and its prognostic implication in Thai CML patients. The objective of this study was to develop a rapid molecular assay for the detection of the two most commonly reported variants of BCR-ABL fusion gene, B2A2 and B3A2 in CML patients. Bone marrow or peripheral blood were used for RNA extraction and reverse-transcribed to cDNA for PCR amplification. 92 per cent of CML patients (91/99) were positive for BCR-ABL gene (61% B3A2 and 31% B2A2). 8/99 CML patients were BCR-ABL-negative. B3A2 and B2A2-positive patients did not have any different clinical and hematological features at presentation although B3A2 patients tended to be slightly older and had higher platelet counts. 71/71 non-CML including other MPD and leukemia cases were all negative for BCR-ABL gene. In conclusion, a rapid RT-PCR assay has now been developed for the detection of this hallmark gene in CML patients. It should be of great value in the differential diagnosis of CML from other diseases. Long-term follow-ups of CML patients with different variants are needed to determine the prognostic importance of each gene variant.
Adult↗
Detection of African horsesickness virus by reverse transcriptase polymerase chain reaction (RT-PCR) using primers for segment 5 (NS1 gene).
The reverse transcription followed by the polymerase chain reaction (RT-PCR) technique was applied to the detection of African horsesickness virus (AHSV) using primers specific for attenuated AHSV serotype 4 segment 5 (NS1 gene). Total RNA which contains both messenger RNA and genomic dsRNA was extracted by the acid guanidinium-phenol-chloroform method from the AHSV infected Vero cells and was used as templates to optimize the RT-PCR. A pair of primer (NP2-NP32) amplified the product of the expected size from all serotypes of attenuated AHSV when four pairs of primers were tested. Using this primer pair, no RT-PCR product was detected from the RNA samples extracted from ten other orbiviruses infected cells and their virions. In addition, RT-PCR using a serial dilution of RNA samples suggested that AHSV was efficiently detected from 1 to 2 cells of the cell monolayer infected with 10(6) TCID50 of AHSV. The RT-PCR concerning with total RNAs of AHSV NS1 gene was found to be a specific and sensitive method for the detection of AHSV.
African Horse Sickness↗
Quantification of vitamin D receptor mRNA in tissue sections demonstrates the relative limitations of in situ-reverse transcriptase-polymerase chain reaction.
In situ-reverse transcriptase-polymerase chain reaction (IS-RT-PCR) is a recently described technique that is used to localize low levels of mRNA within cells and tissue sections. One of the major criticisms levelled at this technique is that positive results may be meaningless, as amplification is required to demonstrate the transcripts of interest. The use of IS-RT-PCR to demonstrate mRNA for receptors for 1,25-dihydroxyvitamin D3 (VDR) in sections of human kidney and bone has previously been described. To ascertain whether the levels of VDR mRNA detected following IS-RT-PCR were transcriptionally significant, computerized image analysis was used to determine the mean silver grain density in human kidney and bone cells following conventional in situ hybridization and after various cycles of IS-RT-PCR. Only a few cycles of PCR were needed to produce an optimum signal, but amplification of signal following IS-RT-PCR was found to be relatively inefficient. Following the optimum number of cycles of IS-RT-PCR in kidney sections, there was a less than four-fold increase in signal. Similarly, in bone, the optimum signal detected was only approximately five times greater than that found with conventional in situ hybridization. These results clearly demonstrate that the increase in signal following IS-RT-PCR follows a more linear pattern and is relatively inefficient, compared with the usual exponential increase with conventional solution phase RT-PCR.
Bone and Bones↗