Combination Of Reverse Transcription Research Articles

Localization of cyclophilin A and cyclophilin C mRNA in murine kidney using RT-PCR

Cyclosporin A (CsA), which is widely used as an immunosuppressant, has a nephrotoxic side effect. The mechanism of this nephrotoxicity is not well understood; however, recent studies suggest that cyclophilin (cyp) is responsible for mediating the immunosuppressive action of CsA through the interaction with the Ca(2+)- and calmodulin-dependent phosphatase, calcineurin. While cyp A mRNA is expressed ubiquitously, cyp C mRNA has been shown to be topically expressed, including in the kidney. We examined: (1) distribution of cyp A and cyp C mRNA in microdissected murine nephron segments, using a combination of reverse transcription and polymerase chain reaction (RT-PCR) techniques, and (2) the effect of CsA administration on cyp C mRNA expression in proximal convoluted tubule. Among the nephron segments examined, large signals for cyp C PCR product were detected in proximal convoluted tubule and proximal straight tubule. Our data showed that the distribution of cyp C mRNA was uneven, and it mainly existed in segments that are relatively sensitive to CsA toxicity. In contrast, cyp A mRNA was found to be distributed almost equally along the nephron segments examined. By CsA administration, the signal for cyp C mRNA PCR product was increased. These results suggest that cyp C may play some role in the renal tubular disorder observed in CsA nephrotoxicity.

Serum hepatitis C virus RNA quantity and histological features of hepatitis C virus carriers with persistently normal ALT levels

We studied hepatitis C virus carriers with normal liver function to evaluate the histological features of their livers and the replicative levels of hepatitis C virus. Liver biopsies were performed in 22 hepatitis C virus carriers with persistently normal ALT levels. Hepatitis C virus RNA in serum was quantified with a competitive assay that combined reverse transcription and the polymerase chain reaction, which is based on co-amplification of the target RNA with known amounts of synthetic mutated RNA. Three patients had normal livers on histological study, whereas the other 19 had chronic persistent hepatitis, with lymphoid infiltrates or aggregates in portal tracts commonly observed but intralobular inflammatory changes absent or minimal. The titer of hepatitis C virus RNA (logarithmic transformed copy number per milliliter of serum) varied from 4.0 to 8.0 (mean +/- S.D.: 6.3 +/- 1.1); it was significantly lower in the three patients with normal livers (4.3 +/- 0.2) than in those with chronic persistent hepatitis with mild (6.4 +/- 0.8, n = 11) or moderate (7.1 +/- 0.5, n = 8) portal inflammation. The titer of hepatitis C virus RNA was correlated with the total score (r = 0.68) and the score for portal inflammation (r = 0.68) in the histological activity index. These results indicated that there seem to be "healthy carriers" of hepatitis C virus with extremely low levels of viral replication. However, in most hepatitis C virus carriers with persistently normal ALT levels, there are inflammatory changes in the portal tracts, with severity depending on the replicative levels of hepatitis C virus.

Long-term carriage of hepatitis C virus with normal aminotransferase after interferon treatment in patients with chronic hepatitis C.

Studies were undertaken to investigate whether interferon therapy could induce hepatitis C virus (HCV) carriage with normal serum alanine aminotransferase (ALT) values using an assay that combined reverse transcription and polymerase chain reaction. The subjects studied were 53 patients with chronic active hepatitis C who received interferon (alpha, 33 cases; beta, 20 cases) therapy. All were seropositive for HCV RNA prior to therapy. In all 22 complete responders, whose ALT levels fell to normal during therapy and for at least 24 weeks after therapy, HCV RNA became persistently negative except in two cases. The two had sustained viremia on treatment and for 1.0-1.5 years of follow-up, although their biochemical tests were normal. In 15 patients with a transient response in whom the disease recurred when interferon was stopped, HCV RNA was undetectable in 80% of the cases at the end of therapy, but the virus reappeared with subsequent elevation of ALT in all patients. However, 3 patients in this group had normal enzyme levels with viremia for 2.1-2.8 years of follow-up after acute deterioration of illness. In 16 patients who did not respond to interferon, HCV RNA was persistently positive during and after therapy. These findings suggest that interferon therapy induces a long-term carrier state of HCV infection with normal ALT levels in some patients.

Quantitative analysis of hepatitis C virus RNA in serum during interferon alfa therapy

Background: Interferon alfa is effective for controlling disease activity in chronic hepatitis C. However, many responders suffer relapse after cessation of therapy. In the present study, the serum concentration of hepatitis C virus RNA was correlated with a sustained response to interferon therapy. Methods: Fifty-three patients with chronic hepatitis C received a 26-week course of interferon alfa. Hepatitis C virus RNA was quantitated in serum at the beginning and end of treatment using a competitive assay that combined reverse transcription and polymerase chain reaction. Results: In long-term responders, whose alanine aminotransferase levels remained within the normal range during the 24 weeks after therapy, the titer of viral RNA (logarithmic transformed copy numbers per milliliter of serum) before therapy (7.1 ± 1.2) was significantly lower (P < 0.001) than that of short-term responders (8.3 ± 0.5) who had a relapse within the 24 weeks after therapy and nonresponders (8.1 ± 0.4). Multivariate multiple logistic regression showed that the titer of viral RNA before therapy was the strongest independent predictor of a sustained response to interferon-alfa therapy (P = 0.002). Conclusions: The titer of hepatitis C virus RNA is the most important factor influencing the sustained response to interferon treatment.

Genic amplification of the entire coding region of the HEF RNA segment of influenza C virus

In order to provide an easy and powerful analysis of influenza C viral HEF RNA segment of a recent strain, a combination of reverse transcription and the polymerase chain reaction was used. We amplified the entire coding region of the HEF gene of a laboratory strain of virus called C/Johannesburg/1/66, widely used for binding and esterase activity studies as well as that of a strain isolated in 1991 (C/Paris/145/91) from a patient suffering from severe flu syndrome. The sequences we amplified were about 2 kilobases long. In this work, we show that the forward ‘universal primer’ Unil, which has been used for influenza A and B viruses cDNA syntheses can also be used for influenza C virus. The PCR primers were designed to contain restriction sites to make the PCR products ready to be used for further purposes. A restriction analysis of the PCR products combined with analyses of all the human influenza C virus HEF gene sequences published so far permitted the design of sets of oligonucleotides which can prime PCR on cDNA of unknown influenza C virus for cloning.

The cDNA sequence, expression pattern and protein characteristics of mouse protein kinase C-ζ

A 2199-bp complementary DNA (cDNA) that encodes protein kinase C-ζ (PKC-ζ) has been isolated from mouse brain by a combination of reverse transcription and primer extension. The predicted PKC-ζ protein consists of 592 amino acids which are 99% identical to those of rat PKC-ζ. Northern blots that were probed with this cDNA revealed abundant 2200-nucleotide (nt) and 4200-nt PKC-ζ mRNAs in mouse brain in roughly equal amounts. PKC-ζ mRNA was also abundant in normal lung, kidney, and testes, and in several hemopoietic tumor lines. In all other mouse tissues and cell lines that were examined, at least faint levels of PKC-ζ mRNAs could also be detected. In tissues other than brain, the amount of PKC-ζ mRNA was less, and the smaller species generally predominated. Furthermore, in these tissues, both PKC-ζ mRNAs appear to be approximately 200 nt longer than the two mRNAs found in the brain. When the cDNA is expressed in insect cells via a baculovirus expression vector, a 75-kDa protein is synthesized which, unlike other PKC isoforms, does not bind phorbol ester, even at very high concentrations.

Detection of endothelin-1 mRNA by RT-PCR in isolated rat renal tubules

A combination of reverse transcription and polymerase chain reaction was utilized to detect baseline endothelin-1 (ET-1) mRNA expressed in renal tubules dissected from rats. 5′ and 3′ primers were constructed according to human ET-1 genomic DNA. Rat ET-1 mRNA was found to be expressed only in cortical through medullary collecting ducts in addition to glomeruli. Sites for tubular synthesis of ET-1 corroborate well with major ET-1 binding sites along the nephron, indicating autocrine/paracrine role of ET-1 in the renal tubules and supporting a prevailing concept on such function of ET-1 in many differing tissues.

Detection of the minus strand of hepatitis C virus RNA by reverse transcription and polymerase chain reaction: implications for hepatitis C virus replication in infected tissue.

The combination of reverse transcription and polymerase chain reaction is a very powerful tool for the detection of hepatitis C virus RNA in sera of patients with hepatitis C virus infection. However, when studying the presence of this virus in tissue using polymerase chain reaction, it may be difficult to distinguish between blood viral particles adhering to the tissue and viral RNA contained within the tissue. Because hepatitis C virus has a single-stranded RNA of positive polarity, a minus-strand RNA is expected to be found in hepatitis C virus-replicating tissues as a template for the synthesis of genomic RNA. To see whether the detection of the minus strand of hepatitis C virus RNA by polymerase chain reaction can be used for the determination of hepatitis C virus-replicating tissues, we examined the presence of the minus strand of hepatitis C virus RNA in the plasma, peripheral blood mononuclear cells and liver specimens of patients with hepatitis C virus infection. The plus-strand RNA was detected in the plasma, peripheral blood mononuclear cells and the liver specimens, but the minus-strand RNA was only detected in the liver. These results suggest that hepatitis C virus replicates in the liver but not in peripheral blood mononuclear cells. This detection method for the minus strand of hepatitis C virus RNA should be useful for determining hepatitis C virus replication in tissues other than liver tissue.

Detection of HIV-1 RNA in heparinized plasma of HIV-1 seropositive individuals

The interference of reverse transcription by heparin was removed by heparinase. When the HIV-1 RNA in the presence of heparin was detected by a combination of reverse transcription and the polymerase chain reaction (PCR), heparinase treatment followed by removal of Ca 2+ before the reverse transcription step permitted the efficient detection of HIV-1 RNA. Prior treatment with heparinase revealed HIV-1 RNA in 68% ( 13 19 ) of heparinized plasma samples from HIV-1 carriers, whereas only 26% ( 5 19 ) of the same specimens were positive without the heparinase step. Heparinase removed the inhibition of reverse transcription by heparin and is highly recommended when detecting low levels of viral RNA in heparinized plasma.

Detection of hepatitis C virus RNA in serum of patients with chronic hepatitis C treated with interferon-alpha.

We tested serial serum samples for hepatitis C virus RNA from patients undergoing treatment for chronic hepatitis C with interferon-alpha using an assay that combined reverse transcription and polymerase chain reaction. The subjects studied were 20 patients with chronic hepatitis who had serum antibody to hepatitis C virus (anti-C100-3). Before therapy, hepatitis C virus RNA was detected in 18 (90%) and 20 (100%) patients using primer sets derived from the NS3 region or the 5'-noncoding region of hepatitis C virus, respectively. Hepatitis C virus RNA became undetectable in all patients whose ALT level fell into the normal range during therapy. However, hepatitis C virus RNA reappeared in all patients whose ALT levels rose again after therapy, usually before the relapse. In patients whose ALT levels did not become normal, hepatitis C virus RNA did not disappear during therapy. Thus therapy with interferon-alpha appears to be beneficial in chronic hepatitis C because of its suppressive effects on hepatitis C virus replication. Detection of hepatitis C virus RNA in serum is useful for evaluating the antiviral effect of interferon.

RT-PCR microlocalization of mRNA for guanylyl cyclase-coupled ANF receptor in rat kidney

Microlocalization of mRNA coding for the guanylyl cyclase-coupled atrial natriuretic factor (ANF) receptor was carried out in the rat kidney. We used a combination of reverse transcription and polymerase chain reaction (RT-PCR) in individual microdissected renal tubule segments, glomeruli, and vasa recta bundles. Relative quantitation of the resulting amplified cDNA utilized densitometry of autoradiograms from Southern blots probed with a specific 32P-labeled probe. Among renal tubule segments, the largest signal was found in the terminal inner medullary collecting duct (IMCD). Slightly smaller signals were found in the initial IMCD and in loop of Henle segments from the inner medulla. Readily detectable signals were also seen in the following segments (in descending order): cortical collecting duct, proximal convoluted tubule, medullary thick ascending limb, cortical thick ascending limb, distal convoluted tubule, and outer medullary collecting duct. Large signals were also detected in glomeruli and in vasa recta bundles from the inner stripe of the outer medulla. Based on these results, we conclude that 1) renal microlocalization of specific mRNAs coding for hormone receptors is feasible through application of the RT-PCR procedure in microdissected renal tubules and vascular elements, and 2) the gene for the guanylyl cyclase-coupled ANF receptor is broadly expressed along the nephron, raising the possibility that multiple sites of ANF action are present.

Detection of tyrosine hydroxylase mRNA and minimal neuroblastoma cells by the reverse transcription-polymerase chain reaction

To facilitate the diagnosis of bone marrow metastasis in neuroblastoma, we have developed a method of amplifying and detecting the tyrosine hydroxylase (TH) mRNA sequence in bone marrow cells using a combination of reverse transcription and the polymerase chain reaction (RT/PCR). By this method, the sequence of TH was detected clearly in the neuroblastoma tissues of all 6 patients and not detected in the bone marrow cells of any of the 9 negative control children. In a reconstitution experiment, 1 neuroblastoma cell per 100 000 normal bone marrow cells could be detected, thus indicating the great sensitivity of this method. Based on these results, this technique may be of value in the diagnosis and treatment follow-up of bone marrow metastasis of neuroblastoma.

Expression and developmental regulation of the k-FGF oncogene in human and murine embryonal carcinoma cells

Embryonal carcinoma (EC) cells provide an effective model system for studying growth factor production and regulation during mammalian embryogenesis. Our earlier data indicated that the mouse EC cell lines F9 and PC-13 and the human EC cell line NT2/D1 produce a factor with properties similar to those ascribed to members of the fibroblast growth factor (FGF) family and that production of this FGF-related factor is suppressed when all three EC cell lines are induced to differentiate. Subsequent studies suggested that NT2/D1 EC cells express transcripts for basic FGF (bFGF). The current study confirms and extends these findings using a combination of reverse transcription and polymerase chain reaction (RT-PCR). In this study, the expression of bFGF and other members of the FGF family have been examined in F9 and PC-13 cells in addition to NT2/D1 EC cells. In contrast to NT2/D1 EC cells, bFGF expression could not be detected in F9 and PC-13 EC cells. Additionally, expression of four other members of the FGF family (acidic FGF, int-2, FGF-5, and FGF-6) were not detected in NT2/D1, F9, or PC-13 EC cells. However, expression of another member of the FGF family, the k-FGF oncogene, was detected in NT2/D1, F9, and PC-13 EC cells. Moreover, the expression of this transcript is reduced dramatically when each of the three EC cell lines is induced to differentiate. Taken together, our findings argue that expression of the k-FGF oncogene is predominantly responsible for the FGF-related activity detected in EC cells and that differentiation of these EC cells results in suppression of this oncogene.

Detection of two alternative bcr/abl mRNA junctions and minimal residual disease in Philadelphia chromosome positive chronic myelogenous leukemia by polymerase chain reaction

The Philadelphia (Ph′) chromosome in chronic myelogenous leukemia (CML) results in fusion of the bcr gene and c-abl oncogene, which transcribes into two types of chimeric bcr/abl mRNAs: the L-6 junction and the K-28 junction. By means of a highly sensitive assay, combination of reverse transcription and polymerase chain reaction (RT/PCR), we analyzed 38 blood samples obtained from 31 patients with Ph′-positive CML and two patients with Ph′-negative bcr rearranged CML. Among the 21 samples obtained in chronic phase, eight patients had the L-6 mRNA, 11 had the K-28 mRNA, and two had both the L-6 and K-28 mRNAs. Among the nine samples obtained in blast crisis, four contained the L-6 mRNA, two contained the K-28 mRNA, and three contained both the K-28 and L-6 mRNAs. This finding supports the concept of alternative splicing of bcr/abl mRNAs transcribed in Ph′-positive CML. However, it appears to be a rare event. Of the eight samples obtained from eight patients who had achieved complete cytogenetic remission and negativity for bcr region rearrangement for 6 months to 3 years after recombinant alpha interferon (r alpha-IFN) therapy, all of them showed evidence of minimal residual Ph′-positive clones as detected by the RT/PCR assay. This finding suggests that interferon therapy suppresses the proliferation of the Ph′-positive clones, but it does not completely eradicate the Ph′-positive stem cells.

Detection of Two Alternative bcr/abl mRNA Junctions and Minimal Residual Disease in Philadelphia Chromosome Positive Chronic Myelogenous Leukemia by Polymerase Chain Reaction

The Philadelphia (Ph') chromosome in chronic myelogenous leukemia (CML) results in fusion of the bcr gene and c-abl oncogene, which transcribes into two types of chimeric bcr/abl mRNAs: the L-6 junction and the K-28 junction. By means of a highly sensitive assay, combination of reverse transcription and polymerase chain reaction (RT/PCR), we analyzed 38 blood samples obtained from 31 patients with Ph'-positive CML and two patients with Ph'-negative bcr rearranged CML. Among the 21 samples obtained in chronic phase, eight patients had the L-6 mRNA, 11 had the K-28 mRNA, and two had both the L-6 and K-28 mRNAs. Among the nine samples obtained in blast crisis, four contained the L-6 mRNA, two contained the K-28 mRNA, and three contained both the K-28 and L-6 mRNAs. This finding supports the concept of alternative splicing of bcr/abl mRNAs transcribed in Ph'-positive CML. However, it appears to be a rare event. Of the eight samples obtained from eight patients who had achieved complete cytogenetic remission and negativity for bcr region rearrangement for 6 months to 3 years after recombinant alpha interferon (r alpha-IFN) therapy, all of them showed evidence of minimal residual Ph'-positive clones as detected by the RT/PCR assay. This finding suggests that interferon therapy suppresses the proliferation of the Ph'-positive clones, but it does not completely eradicate the Ph'-positive stem cells.