Use Of Reverse Transcription-polymerase Chain Reaction Research Articles

Detection and Measurement of Chimeric RNAs by RT-PCR.

Reverse-transcription polymerase chain reaction (RT-PCR) is a powerful combination of assays useful in detection and measurement of expressed RNA transcripts. RT-PCR consists of RNA isolation and reverse transcription into complementary DNA (cDNA), which can then be used as input to a variety of PCR-based procedures such as standard PCR, real-time or quantitative PCR (qPCR or qRT-PCR), or TaqMan PCR procedures. These assays are useful in detection of chimeric transcripts, especially when careful consideration is applied to experimental design. In this chapter, we provide guidelines and procedures for use of RT-PCR in detection and measurement of chimeric RNA expression.

Isolation and detection of duck astrovirus CPH: implications for epidemiology and pathogenicity

ABSTRACTThe transmission routes of duck astrovirus CPH (DAstV/CPH) and its pathogenicity in duck embryos were investigated. Using a reverse transcription-polymerase chain reaction (RT-PCR) developed in this study, DAstV/CPH was detected in 23/50 fresh droppings of breeder ducks, 39/65 breeding eggs, 26/31 dead embryos, and 6/10 newly hatched ducklings, which were taken from a Pekin duck farm where DAstV/CPH had previously been identified. This finding, and the detection of DAstV/CPH in 36/130 dead-in-shell duck embryo samples collected from different hatcheries located in six provinces, suggests that the virus may be horizontally and vertically transmitted and associated with hatchability problems. Inoculation and repeated passages in embryonating duck eggs resulted in isolation of DAstV/CPH. The virus caused severe chorioallantoic membrane lesions as well as growth retardation and embryo mortality, indicating that DAstV/CPH is pathogenic for duck embryos. The effect of DAstV/CPH on hatching was confirmed by an embryo infection experiment in which 8/10 9-day-old duck embryos inoculated with the third passage of DAstV/CPH were unable to hatch, with most embryos succumbing in the final stage of incubation. The use of RT-PCR on the hatched ducklings provided evidence that the embryos could develop into infected ducklings.

Micro-RNA Expression in Hypoplastic Left Heart Syndrome

ObjectiveMicro-RNAs (miRNAs) are important regulators of gene expression through interaction with the 3′UTR of target messenger RNAs (mRNAs). The role of miRNAs has been extensively studied in adult human and nonhuman animal models of heart disease. Hypoplastic left heart syndrome (HLHS) is the most common form of severe congenital heart disease and is an important cause of morbidity and mortality in infants and children. The objective of this work was to analyze the miRNA profile in HLHS patients. Methods and ResultsmiRNA profile was determined in the right ventricle with the use of miRNA array, and expression was validated with the use of reverse-transcription polymerase chain reaction (RT-PCR). Based on bioinformatics analysis, targets were selected and their expression analyzed with the use of RT-PCR.We found that the miRNA profile of HLHS is novel, with few similarities between pediatric and adult idiopathic dilated cardiomyopathy. Moreover, our analysis identified putative targets for these miRNAs that are known to be important for cardiac development and disease, and that miRNAs and their putative targets are antithetically regulated. We also found that miRNA expression changes with stage of surgery, suggesting that volume unloading of the ventricle has important consequences for gene expression. ConclusionsOur data suggest a unique miRNA profile for HLHS that may be associated with defects in cardiac development and disease.

СИНДРОМ БОЛЬШОЙ ПЕЧЕНИ И СЕЛЕЗЕНКИ — НОВАЯ ВИРУСНАЯ БОЛЕЗНЬ КУР В РОССИИ

For the first time in Russia, the viral agent of hepatitis splenomegaly syndrome, a disease of laying hens from broilers’ parental flock, has been identified by the use of RT-PCR (reverse transcription — polymerase chain reaction). On a poultry farm a low laying ability and high death of sick birds were examined, and the dissection revealed the bloody liquid in abdominal cavity, the hepatoand splenomegaly, the hepatic subcapsular hematomas and a breakable consistence of the liver. The viruses that cause similar clinical symptoms (adenovirus, Marek’s disease virus, leucosis virus) were not found. The sequence analysis of DNA fragment of ORF2 capsid protein gene of isolated virus and subsequent comparison with analogous sequences from GenBank database (NCBI) confirmed the identity of the agent to hepatitis E virus. Amplicon sequencing showed 61.6-78.6 % homology to the sequences from GenBank for capsid protein ORF2 gene of hepatitis E virus.

Application of RT-PCR for diagnosis of respiratory syncytial virus and human metapneumovirus infections in Bulgaria, 2006-7 and 2007-8

We describe here the results of respiratory syncytial virus (RSV) detection by reverse transcription polymerase chain reaction (RT-PCR) during two consecutive seasons, from December 2006 to February 2007 and from October 2007 to March 2008, performed in the National Laboratory of Influenza and Acute Respiratory Diseases, Bulgaria. A total number of 278 nasopharyngeal samples obtained from hospitalised children up to the age of five years were investigated for these two seasons. During the first season, the aetiological role of RSV was confirmed in 56 of 148 samples (37.8%) compared to 11 of 130 samples (8.5%) during the second season. Since the beginning of January 2008, RT-PCR for the detection of the recently identified human metapneumovirus (HMPV) has also been introduced in Bulgaria. This virus has been demonstrated as the aetiological agent in 13 out of 81 samples (16%) from children of the same age group. The use of RT-PCR allows the detection of a broader spectrum of viruses causing respiratory diseases, as well as better discrimination of the aetiological agents in clinically similar cases.

Broad-spectrum detection and quantitation methods of Soil-borne cereal mosaic virus isolates

A broad-spectrum reverse transcription-polymerase chain reaction (RT-PCR) protocol was developed for detecting Soil-borne cereal mosaic virus (SBCMV) isolates, responsible for mosaic diseases in Europe, using primers targeting the highly conserved 3′-untranslated region of RNA-1 and RNA-2 of SBCMV. The 3′-end region is a privileged target for the detection of a wide range of isolates, because of sequence conservation, of the tRNA-like structure, the major role in viral replication and the signal amplification due to the presence of numerous genomic and subgenomic RNAs. The primers were also designed for virus quantitation using real-time RT-PCR with SYBR-Green chemistry. No cross-reaction with Wheat spindle streak mosaic virus, frequently associated with SBCMV, was observed. The use of RT-PCR and real-time quantitative RT-PCR allowed a more sensitive detection and quantitation of SBCMV to be made than was the case with ELISA. The methods enabled European isolates of SBCMV from Belgium, France, Germany, Italy and the UK to be detected and quantified. Real-time RT-PCR represents a new tool for comparing soil inoculum potential as well as cultivar resistance to SBCMV.

Reverse Transcription-Polymerase Chain Reaction on Pooled Samples to Detect Bovine Viral Diarrhea Virus by using Fresh Ear-Notch—Sample Supernatants

Ear-notch samples from 3,599 yearling heifers were collected to detect persistently infected (PI) animals with suspect bovine viral diarrhea virus (BVDV). Individual immunohistochemistry (IHC), individual antigen-capture enzyme-linked immmunosorbent assay (AC-ELISA), and reverse transcription-polymerase chain reaction (RT-PCR) tests with pooled ear-notch supernatants were compared with samples from 3,016 heifers, whereas RT-PCR ear-notch pools and individual AC-ELISA tests were compared with samples from all 3,599 heifers. Four heifers were identified positive by both IHC and AC-ELISA, whereas the remaining heifers were identified negative by both tests. When supernatant from ear notches from 100 animals was randomly pooled and RT-PCR was accomplished on each pool, RT-PCR identified 2 pools that contained 1 positive AC-ELISA sample and 1 pool that contained 2 positive AC-ELISA samples. Further evaluation of the pooled RT-PCR ear-notch supernatant detected 100% (n = 36) samples spiked with supernatant from a single randomly selected positive AC-ELISA ear notch. Although follow-up confirmatory tests were not completed, all 3 methods correlated 100% in detecting suspect PI animals, with a kappa value of 1. The use of RT-PCR on pooled ear-notch supernatant could provide an initial, rapid, cost-effective method of screening cattle herds for BVDV PI animals. Subsequent serial testing with an AC-ELISA to evaluate individual samples included in the positive pool could minimize the length of time other animals are exposed to the virus.

A reverse transcription-PCR assay to detect viable Mycoplasma synoviae in poultry environmental samples

In order to study horizontal transmission of Mycoplasma synoviae an avian pathogen, a reverse transcription-polymerase chain reaction (RT-PCR) assay was developed to detect viable Mycoplasma in environment. The test was based on the RT-PCR of the 16S ribosomal RNA (rRNA) of Mycoplasma genus. Results showed that Mycoplasma 16S rRNA was stable up to 23 h after cell death. Therefore, the test allowed detection of viable or very recently (less than 23 h) dead mycoplasmas. M. synoviae survival in artificially contaminated water, food and soil and in the environment of M. synoviae experimentally infected turkeys was estimated by culture and RT-PCR. The RT-PCR method was then applied in a naturally infected laying hen farm showing problems of recurrent mycoplasmosis in the hens. Results confirmed the usefulness of RT-PCR in checking the efficiency of biosecurity measures and in improving cleaning and disinfection protocols.

Persistent measles virus infection and otosclerosis.

The cause of otosclerosis is still unknown. Recently, measles virus involvement has been implicated. The aim of this study was to analyze the presence of measles virus RNA within the otosclerotic focus and to evaluate the perilymphatic antibody pattern. Bone and perilymph specimens from 40 patients with the spontaneous form of otosclerosis and from control patients were investigated by reverse transcription polymerase chain reaction (RT-PCR), Western blot techniques, and cell culture. By the use of RT-PCR, measles virus RNA could be detected in 32 patients, but not in controls. Analysis of perilymph revealed the presence of antibodies to N, F1, and M measles virus proteins in all cases, and antibodies against H protein in 2 additional cases. In preosteoblasts cultured from otosclerotic bone chips, no measles virus RNA could be amplified. We conclude that the spontaneous form of otosclerosis is, in the vast majority of cases, a measles virus-associated disease of the otic capsule.

Detection of Viable Vibrio mimicus by Reverse Transcription-Polymerase Chain Reaction.

Differentiating viable cells from nonviable cells is of considerable importance in the monitoring of food-borne pathogens. A reverse transcription-polymerase chain reaction (RT-PCR) assay was developed to detect mRNA from the phospholipase gene (phl) of Vibrio mimicus. Viable V. mimicus cells were killed by heat or ethanol treatment and kept for various periods at room temperature. Total RNA from V. mimicus was extracted, treated with DNase and subjected to RT-PCR with primers for the phl gene. The phl mRNA was detected in the viable cells, but it gradually disappeared when the killed cells were left at room temperature and became undetectable after 8 h of storage. Furthermore, RT-PCR generated a 493 by fragment from the total RNA extracted from as few as about 103 organisms, confirming the sensitivity of the assay. The amplification of the phl mRNA was specific for V. mimicus, as no amplification was found when fifteen other Vibrio species and seven related organisms were tested. The results indicated a good relationship between the detection of the phl mRNA and viability of V. mimicus cells because the phl transcript is rapidly degraded upon cell death. This work shows the usefulness of RT-PCR as a sensitive method for the specific detection of viable V. mimicus.

Reverse-transcription polymerase chain reaction detection of the enteroviruses.

c Objective.—This review focuses on commercial and inhouse‐developed reverse-transcription polymerase chain reaction (RT-PCR) assays used for the detection of enteroviral infections. In addition to providing details on the performance of RT-PCR, its specificity, and sensitivity, the clinical utility of this diagnostic method with specific reference to its impact on hospitalization and cost savings is addressed. Data Sources.—MEDLINE was searched for reports relating to RT-PCR detection of the enteroviruses in adults and children. The search was restricted to studies reported in English language journals. Study Selection.—Reports documenting detailed information regarding the RT-PCR conditions, primers, sensitivity, specificity and, if relevant, clinical impact were selected for analysis. Data Extraction.—Details regarding method of extraction of the enteroviral genome, the primers used, RT-PCR conditions, and sensitivity and specificity of the assay were extracted from the literature. For reports detailing the use of RT-PCR in the clinical management of enteroviral infections in children, the reduction in duration of hospitalization and health care cost savings were recorded. Data Synthesis.—Reverse-transcription PCR can increase the yield of detection of enteroviruses from cerebrospinal fluid by a mean of approximately 20% over tissue culture. Reverse-transcription PCR of cerebrospinal fluid has been shown to exhibit sensitivity and specificity values of 86% to 100% and 92% to 100%, respectively. Reductions of 1 to 3 days of hospitalization per patient are predicted if RTPCR is used to diagnose enteroviral meningitis in children. Conclusions.—Reverse-transcription PCR detection of enteroviral infections is an extremely rapid, sensitive, and specific diagnostic modality. Both commercial assays and assays developed in-house appear to be equivalent with regard to sensitivity and specificity. Reverse-transcription PCR diagnosis of enteroviral infections in children could reduce the length of hospitalization and result in significant health care cost savings. (Arch Pathol Lab Med. 1999;123:1161‐1169)

Reverse-transcription polymerase chain reaction detection of the enteroviruses.

This review focuses on commercial and in-house-developed reverse-transcription polymerase chain reaction (RT-PCR) assays used for the detection of enteroviral infections. In addition to providing details on the performance of RT-PCR, its specificity, and sensitivity, the clinical utility of this diagnostic method with specific reference to its impact on hospitalization and cost savings is addressed. MEDLINE was searched for reports relating to RT-PCR detection of the enteroviruses in adults and children. The search was restricted to studies reported in English language journals. Reports documenting detailed information regarding the RT-PCR conditions, primers, sensitivity, specificity and, if relevant, clinical impact were selected for analysis. Details regarding method of extraction of the enteroviral genome, the primers used, RT-PCR conditions, and sensitivity and specificity of the assay were extracted from the literature. For reports detailing the use of RT-PCR in the clinical management of enteroviral infections in children, the reduction in duration of hospitalization and health care cost savings were recorded. Reverse-transcription PCR can increase the yield of detection of enteroviruses from cerebrospinal fluid by a mean of approximately 20% over tissue culture. Reverse-transcription PCR of cerebrospinal fluid has been shown to exhibit sensitivity and specificity values of 86% to 100% and 92% to 100%, respectively. Reductions of 1 to 3 days of hospitalization per patient are predicted if RT-PCR is used to diagnose enteroviral meningitis in children. Reverse-transcription PCR detection of enteroviral infections is an extremely rapid, sensitive, and specific diagnostic modality. Both commercial assays and assays developed in-house appear to be equivalent with regard to sensitivity and specificity. Reverse-transcription PCR diagnosis of enteroviral infections in children could reduce the length of hospitalization and result in significant health care cost savings.

Detection of Salmonella enteritidis by reverse transcription-polymerase chain reaction (PCR)

A reverse transcription-polymerase chain reaction (RT-PCR) method was developed for detecting mRNA from the sefA gene of Salmonella enteritidis. Detection of target mRNA was examined from cells grown in buffered peptone water at different temperatures (37, 25 and 15°C) and pH (5.5, 7.2 and 8.5). The results revealed that the levels of transcription of the sefA gene differed depending upon the physiological state of the cells. This affected the sensitivity of the RT-PCR assay. When the assay was evaluated for the detection of S. enteritidis PT4 in artificially contaminated minced beef and whole egg samples, an enrichment step was used (buffered peptone water, pH 7.2, 37°C, 16 h) to increase the sensitivity of the assay. In the presence of the normal background flora of each food type, it was possible to detect ten cells of S. enteritidis PT4 after a 16-h enrichment using the RT-PCR assay, with a total testing time of 28 h. Unlike the PCR test for the sefA gene that was tested in parallel, the RT-PCR assay did not detect nonviable (heat-inactivated) S. enteritidis PT4 cells. The results supported the usefulness of RT-PCR as a method for the detection of viable microorganisms.

Application of electronmicroscopy, enzyme immunoassay, and RT-PCR to monitor an outbreak of astrovirus type 1 in a paediatric bone marrow transplant unit.

During 1997, an extensive outbreak of astrovirus occurred in a unit where paediatric patients were being treated for leukaemias and inherited immune deficiency disorders. Prolonged shedding of virus for many months following infection was demonstrated in three patients who had undergone bone marrow transplantation. Comparison of reverse transcription-polymerase chain reaction (RT-PCR), enzyme immunoassay (EIA), and electronmicroscopy (EM) to monitor the outbreak showed that many subclinical infections, mainly in children aged > 3 years could only be detected by RT-PCR. Use of RT-PCR revealed that several patients were infected earlier and shed virus for longer than by using EM or EIA. The virus responsible for the outbreak was identified as HAstV-1 and was shown to have a sequence that differed from a strain obtained in 1988.

Detection of disseminated prostate cells by reverse transcription-polymerase chain reaction (RT-PCR): technical and clinical aspects.

Metastasis of prostate cancer (CaP) to bone is responsible for much of the morbidity and mortality associated with this disease. While the primary goal of current methods of treatment is to prevent, slow down or reverse metastatic spread, the sensitivity of today’s conventional staging techniques is insufficient to detect micrometastatic disease. Radical prostatectomy is intended to be a complete cure, but 15–30% of these cases recur, because CaP cells that have escaped the prostate prior to surgery give rise to metastases. Although macroscopic techniques such as the bone scan cannot detect micrometastases, new molecular approaches that are under development may prove to be capable of filling this need. There is now hope that detection of prostate cells in circulation may provide a new and highly sensitive staging tool. Because prostate-specific antigen (PSA) is produced almost exclusively by prostate epithelial cells (Clements and Mukhtar, 1994; Levesque et al., 1995a,b; Oesterling, 1991; Zarghami and Diamandis, 1996; Zarghami et al., 1997), the presence of PSA-positive cells in circulation clearly indicates an abnormal condition, and a strong correlation between the appearance of prostate cells in circulation and the advent of metastasis might be expected. Current models of metastasis hold that cells ‘‘shed’’ by primary tumors are borne by the circulation (blood and lymphatics) to other organs, where they are deposited and form secondary tumors. It is not known whether these circulating cells have metastatic potential, or whether they must undergo further changes to cause metastases. Protocols based on the reverse transcription-polymerase chain reaction (RT-PCR) have been developed in response to the need for identification and widespread adoption of superior methods for detecting these extra-prostatic cells, so that more systematic studies with long-term follow-up may be undertaken. Currently, the mRNAs encoding PSA, prostate-specific membrane antigen (PSMA), and human glandular kallikrein (hK2) are the 3 messages most frequently used for detection of prostate cells, but these messages, while prostate-specific, are not cancer-specific, and therefore yield little or no information about the metastatic potential of the cells detected. Markers related to cancer progression are desired from the viewpoints of both prognostic methods and basic understanding. The limit of detection (LoD) of RT-PCR is better than that of other methods in current use, such as flow cytometry and immunohistochemistry (Pelkey et al., 1996). There is therefore considerable interest in the use of RT-PCR for detection and possible quantification of prostate cells in peripheral blood (PB), bone marrow (BM) and lymph node (LN) samples from CaP patients. However, despite the promise of this method, it is not yet clear whether RT-PCR will prove to be of clinical value in prostatecancer staging. In this review we will summarize published reports on RT-PCR detection of circulating prostate cells. Because the field is developing rapidly, there have been several significant advances since the last major reviews were published (Gomella et al., 1997; Olsson et al., 1997), including the use of longer primers with 2-temperature PCR for specificity, development of internal controls and targeting of the hK2 message. There have also been a number of additional reports describing RT-PCR protocols and clinical results in CaP. With a very few exceptions, we have restricted the scope of this review to published articles. The review is divided into 2 parts: a technical evaluation of RT-PCR assays used for detection of prostate cells and a discussion of the results of clinical studies using these procedures.

Expression of Extracellular Calcium (Ca2+o)-Sensing Receptor in Human Peripheral Blood Monocytes

The calcium-sensing receptor (CaR) is a G protein-coupled receptor playing key roles in extracellular calcium ion (Ca2+o) homeostasis in parathyroid gland and kidney. Macrophage-like mononuclear cells appear at sites of osteoclastic bone resorption during bone turnover and may play a role in the “reversal” phase of skeletal remodeling that follows osteoclastic resorption and precedes osteoblastic bone formation. Bone resorption produces substantial local increases in Ca2+othat could provide a signal for such mononuclear cells present locally within the bone marrow microenvironment. Indeed, previous studies by other investigators have shown that raising Ca2+oeitherin vivoorin vitrostimulated the release of interleukin-6 (IL-6) from human peripheral blood monocytes, suggesting that these cells express a Ca2+o-sensing mechanism. In these earlier studies, however, the use of reverse transcription-polymerase chain reaction (RT-PCR) failed to detect transcripts for the CaR previously cloned from parathyroid and kidney in peripheral blood monocytes. Since we recently found that non-specific esterase-positive, putative monocytes isolated from murine bone marrow express the CaR, we reevaluated the expression of this receptor in human peripheral blood monocytes. Immunocytochemistry, flow cytometry, and Western blot analysis, performed using a polyclonal antiserum specific for the CaR, detected CaR protein in human monocytes. In addition, the use of RT-PCR with CaR-specific primers, followed by nucleotide sequencing of the amplified products, identified CaR transcripts in the cells. Therefore, taken together, our data show that human peripheral blood monocytes possess both CaR protein and mRNA very similar if not identical to those expressed in parathyroid and kidney that could mediate the previously described, direct effects of Ca2+oon these cells. Furthermore, since mononuclear cells isolated from bone marrow also express the CaR, the latter might play some role in the “reversal” phase of bone remodeling, sensing local changes in Ca2+oresulting from osteoclastic bone resorption and secreting osteotropic cytokines or performing other Ca2+o-regulated functions that contribute to the control of bone turnover.

Reverse transcription polymerase chain reaction is a reliable assay for detecting leukemic colonies generated by chronic myelogenous leukemia cells.

Single-colony karyotyping (SCK) and reverse-transcription polymerase chain reaction (RT-PCR) are two increasingly used techniques for the quantification of leukemic colonies generated by chronic myelogenous leukemia (CML) cell fractions purged or selected in vitro. Recently, the existence of Philadelphia (Ph) chromosome positive progenitors with a silent BCR-ABL gene has been reported, thus raising concerns on the use of RT-PCR for detecting BCR-ABL positive progenitors. In order to investigate this issue further, colonies (n = 204) generated by mononuclear (MNC) or CD34+ CML cells were individually harvested, divided into two aliquots and analyzed both at the cytogenetic level to detect the Ph chromosome, and the molecular level to detect BCR-ABL transcripts. The mean (+/- s.d.) percentages of colonies analyzable by either SCK or RT-PCR were 74 +/- 16% and 86 +/- 16%, respectively. A significant percentage of colonies (67 +/- 19%) could be successfully analyzed by both SCK and RT-PCR. Although the majority of these colonies (97 +/- 5%) were Ph-positive and BCR-ABL-positive, a negligible percentage (4%) of progenitors were Ph-positive but BCR-ABL-negative. In order to test the influence of colony size on the outcome of molecular analysis, the efficiency of our RT-PCR assay in detecting BCR-ABL transcripts was investigated by means of experiments in which the number of cells used to start RNA extraction was serially reduced. These experiments showed that at least 150 cells were necessary to achieve a reproducible amplification of BCR-ABL transcripts. By correlating the size of harvested colonies with the outcome of molecular analysis, it was evident that BCR-ABL-negative but Ph-positive colonies represented false negative results occurring when a number of leukemic cells below the detection limit of our RT-PCR assay was analyzed. In conclusion, our data demonstrate that individual CML colonies grown in semisolid culture assays can be indifferently analyzed by SCK or RT-PCR, and support an extensive use of a carefully standardized RT-PCR assay to estimate the leukemic burden within samples which have been purged and selected in vitro.

Gastrin inhibits secretin-induced ductal secretion by interaction with specific receptors on rat cholangiocytes.

We assessed the effect of gastrin on ductal secretion in normal and bile duct-ligated (BDL) rats. The effect of gastrin on ductal secretion was examined in the presence of proglumide, a specific antagonist for gastrin receptor (GR). We isolated pure cholangiocytes from normal and BDL rats and assessed gastrin effects on secretin receptor (SR) gene expression and intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels. We examined the presence of GR mRNA in cholangiocytes by reverse transcription polymerase chain reaction (RT-PCR). In normal or BDL rats, gastrin produced no changes in spontaneous bile secretion. Simultaneous infusion of gastrin inhibited secretin-induced choleresis and bicarbonate output in BDL rats. In the presence of proglumide gastrin did not inhibit secretin-induced choleresis in BDL rats. Gastrin decreased in cholangiocytes from BDL rats 1) SR gene expression and 2) secretin-induced cAMP levels. With the use of RT-PCR, GR mRNA was detected in cholangiocytes. Similar to what is shown for secretin and somatostatin, we propose that the opposing effects of secretin and gastrin on cholangiocyte secretory activity regulate ductal secretion in rats.

A reverse transcription-polymerase chain reaction (RT-PCR) approach for cloning Ah receptors from diverse vertebrate species: Partial sequence of an Ah receptor from the teleost Fundulus heteroclitus

The aromatic hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates many of the biological effects of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) and related halogenated aromatic hydrocarbons. AhR proteins are expressed in most vertebrate animals, including teleost and elasmobranch fish, but the relationship between piscine and mammalian Ah receptors is uncertain. In the present studies, cloning of an Ah receptor cDNA from the teleost Fundulus heteroclitus was pursued using a reverse transcription-polymerase chain reaction (RT-PCR) approach. Degenerate oligodeoxynucleotides were designed based on conserved regions of mammalian Ah receptors and the related PAS proteins Per, ARNT and Sim. The oligonucleotides were used as primers in the RT-PCR to amplify a portion of an AhR expressed in Fundulus liver. A fragment of approximately 700 bp was amplified and directly sequenced. The deduced amino acid sequence of this fragment (212 residues) shares significant sequence identity with the PAS domains of the mouse (64%), rat (64%) and human (62%) Ah receptors. These data reveal conservation of Ah receptor structure between mammals and fish, vertebrate groups separated by over 400 million years of evolution. The successful use of RT-PCR to amplify a Fundulus AhR establishes the utility of this approach in the phylogenetic analysis of Ah receptor structure and function.

Analysis of plant gene expression by reverse transcription-PCR.

As described in previous chapters ( Chapters 15– 23), techniques for the measurement of gene expression at the RNA level include RNA gel blot analysis, S1 mapping, primer extension, RNase protection, and, more recently, reverse transcription-polymerase chain reaction (RT-PCR) (). This involves the reverse transcription of total RNA (or mRNA) from which cDNA is synthesized. Primers designed to the target cDNA are then used in a PCR reaction to amplify the desired product. This is then identified by the predicted size of the cDNA on agarose gels with size markers. The identity can also be confirmed by DNA gel blot analysis with a homologous probe or by the use of an end-labeled primer in the PCR reaction from which autoradiographs of gels run with end-labeled size markers that give both a sensitive and accurate determination of product (). This is necessitated when PCR conditions are suboptimal and result in low concentrations of product nonvisible in ethidium bromide stained gels. However, as PCR is a commonly used technique, the need to optimize should not act as a deterrent to the use of RT-PCR. The advantages of RT-PCR include its simplicity in comparison to techniques such as RNase protection and the ability to detect rare transcripts or, similarly, mRNA from small tissue samples. Problems associated with RT-PCR, when the detection of a transcript from transgenic plants is complicated by cDNA copies in genomic DNA contamination of RNA, are discussed in Note 3.