Reverse Transcriptase-polymerase Chain Reaction System Research Articles

Endogenous Retrovirus Erv-3 Is Not Implicated in Rheumatoid Arthritis But May Provide a Biomarker for Osteoarthritis: Table 1

Rheumatoid arthritis (RA) is an autoimmune disease with chronic inflammation of the synovial membrane culminating in the destruction of cartilage and bone. The etiology is unknown, although possible causes include viral triggers1. The latter may include human endogenous retroviral (HERV) families: HERV-K, HERV-L, ERV-9, and ERV-32,3. We have reported a novel reverse transcriptase polymerase chain reaction (RT-PCR) system to identify HERV-K10 and identified significantly high levels of messenger RNA in the peripheral blood (and synovial fluid) of patients with RA compared to osteoarthritis (OA) and healthy controls4. In our study, we modified the RT-PCR system to analyze ERV-3 expression in these patients. Peripheral blood mononuclear cells (PBMC) from 20 RA patients who fulfilled American Rheumatism Association criteria were used to extract … Address correspondence to Dr. P. Nelson; E-mail: p.n.nelson{at}wlv.ac.uk

Microfluidic Chips for Detecting the t(4;14) Translocation and Monitoring Disease during Treatment Using Reverse Transcriptase-Polymerase Chain Reaction Analysis of IgH-MMSET Hybrid Transcripts

Diagnosis platforms incorporating low-cost microfluidic chips enable sensitive, rapid, and accurate genetic analysis that could facilitate customized therapies tailored to match the vulnerabilities of any types of cancer. Using ex vivo cancer cells, we have detected the unique molecular signature and a chromosomal translocation in multiple myeloma. Multiple myeloma is characterized by IgH rearrangements and translocations that enable unequivocal identification of malignant cells, detected here with integrated microfluidic chips incorporating genetic amplification via reverse transcriptase-polymerase chain reaction and capillary electrophoresis. On microfluidic chips, we demonstrated accurate and versatile detection of molecular signatures in individual cancer cells, with value for monitoring response to therapy, detecting residual cancer cells that mediate relapse, and evaluating prognosis. Thus, testing for two clinically important molecular biomarkers, the IgH VDJ signature and hybrid transcripts signaling the t(4;14) chro-mosomal translocation, with predictive value in diagnosis, treatment decisions, and monitoring has been efficiently implemented on a miniaturized microfluidic system.

Expression of ice, bcl-2, c-myc and p53 in different bone marrow cell populations from patients with diffuse large B-cell lymphoma

We have previously reported functional alterations in vitro in the hematopoietic compartment of patients with diffuse large B-cell lymphoma (DLBCL). In the present study, we assessed the presence of molecular alterations in hematopoietic cells derived from DLBCL marrow. Accordingly, the expression of four genes (i.e. ice, bcl-2, c-myc and p53) was assessed both, at the mRNA and protein levels, in three cell populations: (i) population I, consisting of morphologically recognizable precursor and mature cells; (ii) population II, enriched for CD34+ Lineage-negative (Lin−) cells; and (iii) population III, enriched for CD34+ CD38− Lin− cells. By using a multiplex reverse transcriptase-polymerase chain reaction system, we observed reduced expression of bcl-2 in population I, and c-myc in populations I and II from lymphoma marrow compared to their normal counterparts. On the other hand, expression of ice and p53 was not significantly different when comparing normal and DLBCL samples. At the protein level, all four molecules were expressed in a higher proportion of samples from DLBCL patients than in marrow samples from normal subjects. Interestingly, these proteins were expressed predominantly in primitive cells (population III), whereas the proportion of positive samples was reduced in population II, and even more in population I. Taken together, our results indicate that, in DLBCL, molecular alterations are present in hematopoietic cells from bone marrow, including morphologically recognizable precursor and mature cells, as well as primitive hematopoietic progenitors (CD34+ cells). To date, the physiological implications of these alterations are still unclear, and further studies should be undertaken to address this issue.

Low copy number detection of herpes simplex virus type 1 mRNA and mouse Th1 type cytokine mRNAs by Light Cycler quantitative real-time PCR

The real-time principle of reverse transcriptase polymerase chain reaction (RT-PCR) provides a quantitative and reproducible method to detect low copy number transcripts. The quantitative detection of cytokines from tissue samples is complicated by the low expression rates and the short half-lives of the cytokine proteins. The methods have been insensitive and labor-intensive. The LightCycler technique provides a 30-min PCR system with continuous fluorescent detection, analysis of the melting points of the products and user-friendly software for the analysis of the unknown samples. External copy number standards enable the measurement of amounts of the desired targets. We demonstrate the dynamic range of the RT-PCR system from a 100 to 10 7 mRNA copies of the mouse Th1 cytokines interleukin- (IL-) 12p35, 12p40 and IL-23p19 as well as gamma interferon (IFN-γ) and the housekeeping gene β-actin, with the usage of fluorescent hybridization probes. The cytokine quantitation was exemplified in murine nervous system samples. A viral transcript, mRNA of alpha trans-inducing factor (αTIF), or VP16 gene, of herpes simplex virus (HSV) type 1 was used to quantitate the viral replication in infected cells and in murine nervous system samples. For this viral transcript the linear dynamic range spanned from ten copies to one million copies (highest tested). For all tested cytokine transcripts, the detection level with the dsDNA binding dye SYBR Green I was one log lower than with the hybridizing fluorescent probes. The viral transcript was detected even with the SYBR Green I system at the level of ten copies. The specificity of the PCR was reached with the use of TaqStart antibody, by careful design of primers and probes, by melting temperature analysis and comparison with the gel electrophoresis and Southern blot analysis.

Use of an internal standard in a closed one-tube RT-PCR for the detection of equine arteritis virus RNA with fluorescent probes.

Routine detection of equine arteritis virus (EAV) can be achieved by virus isolation (VI) in cell culture, or by the amplification of viral genome by molecular methods. To simplify molecular diagnosis, a number of different Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and RT-nested PCR (RT-nPCR) assays were compared, and a one-tube method was developed and optimised utilizing a fluorogenic probe (TaqMan). An artificial RNA template (Mimic) and associated probe were also constructed to provide in-tube validation of the RT-PCR system. To assess the utility of the RT-PCR TaqMan assay, 28 different isolates of EAV representing different genetic groups of American and European strains were tested. Furthermore, the ability of VI and RT-PCR TaqMan assay to detect EAV in different biological matrices such as semen, nasal and faecal swabs and blood was compared. All 28 EAV strains were detected by the RT-PCR TaqMan assay. The results of TaqMan and VI testing were in agreement for 30 of the 33 semen samples and all of the 50 other clinical specimens examined: the RT-PCR TaqMan assay detected 18 positive semen samples, three more than VI. In conclusion, the one-tube RT-PCR TaqMan assay is a rapid, reliable method for the detection of EAV.

Development of a multiplex real-time reverse transcriptase-polymerase chain reaction for equine infectious anemia virus (EIAV)

A single-tube reverse transcriptase-polymerase chain reaction (RT-PCR) using a fluorogenic real-time PCR detection method is described for the quantitation of equine infectious anemia virus (EIAV) RNA in the plasma of equids. To compensate for variations inherent in sample preparation a multiplex real-time RT-PCR system was developed that permitted the simultaneous calculation of the nucleic acid recovery rate along with the copy number of viral RNA molecules. Detection of EIAV RNA was linear from 10 9 to 10 1 molecules with intra- and inter-assay variability of less than 1% at 10 8, 10 6, 10 4 and 10 2 molecules.

PCR detection of circulating tumor cells in nasopharyngeal carcinoma patients with distant metastasis: effect of enzyme and sampling.

Nasopharyngeal carcinoma (NPC) has a high potential to develop distant metastasis after radiotherapy. Cytokeratin 19 (CK-19) mRNA has been frequently used as a marker in the detection of circulating tumor cells of epithelial origin, but has rarely been investigated in NPC. This study was performed to evaluate the effect of blood sampling and different Taq DNA polymerase on the results of nested reverse transcriptase-polymerase chain reaction (RT-PCR) assay. Peripheral blood samples from a total of 37 NPC patients with well-documented distant metastasis (M1) were collected before treatment. Eighteen patients had more than one blood sampling. Five different Taq DNA polymerases were used to test the blood from 17 patients. Peripheral blood of 37 nonmetastatic (M0) NPC patients was tested by the same nested RT-PCR system using multiple Taq DNA polymerases to evaluate the impact of multienzyme assay in the prediction of subsequent distant metastasis. Among M1 NPC patients, the accumulative positive rates of CK-19 mRNA were 22.2%, 44.4%, 70.6%, 75.0%, and 80.0% when one, two, three, four, or five blood sampling were taken, respectively. The accumulative positive rates increased as the numbers of different enzymes increased-from 35.5% by one enzyme to 82.4% by five enzymes. Six of 37 M0 patients had distant metastasis develop after a median follow-up time of 20 months. The detection sensitivity for four-enzyme test (5 of 6 = 83.3%) is better than that of one-enzyme test (2 of 6 = 33.3%). Our data demonstrate that multiple blood sampling or using multiple enzymes for nested RT-PCR assay significantly enhances the sensitivity in the molecular diagnosis of NPC metastasis.

Development of a fluorogenic RT-PCR system for quantitative identification of dengue virus serotypes 1–4 using conserved and serotype-specific 3′ noncoding sequences

A fluorogenic reverse transcriptase-polymerase chain reaction (RT-PCR) system was developed for use as a rapid diagnostic test for determining dengue viremia. The dengue virus 3′-noncoding sequence was utilized to formulate serotype-specific RT-PCR assays for quantitative identification of the four different dengue virus serotypes. A generic RT primer set containing two dengue specific anti-sense primers (DV-L1 and DV-L2) could be used to transcribe extracted viral RNA of all four dengue virus types to complimentary DNA (cDNA). The resultant dengue viral cDNA could be quantitatively identified at the serotype level by the 5′–3′ exonuclease assay using four serotype-specific sense primers. The fluorogenic dengue type-specific RT-PCR can detect each of the four dengue types at similar low detection limits, i.e. 20–50 plaque forming units per milliliter of serum. Two panels with four dengue reference serotypes and 134 clinical samples were used to validate detection sensitivity and specificity of the dengue serotype RT-PCR assay, using virus isolation in cell culture as the criterion standard. By analyzing sera samples from Puerto Rico that were collected from 1999 through 2000, the assay demonstrated high level detection sensitivity and specificity of 92.8 and 92.4%, respectively, for all four dengue virus serotypes.

Absolute quantification of human chorionic gonadotropin-beta mRNA with TaqMan detection. 4.

We describe a reverse transcriptase-polymerase chain reaction (RT-PCR) for determination of human chorionic gonadotropin-beta (HCG beta) mRNA copies using the TaqMan system. To evaluate our quantitative assay, we analyzed HCG beta transcripts of all protein coding genes (HCG beta 5, 3, 8, and 7) in human RNA panels of different normal tissues and in glycodelin-A-stimulated trophoblast cell cultures. Absolute quantification using HCG beta TaqMan probe was found to be highly reproducible. Our study of RNA panels confirms recently published results that expression of HCG beta transcripts is a common feature of a great variety of different normal tissues. High levels of HCG beta mRNAs (> 1,000 molecules per 200 ng RNA) were detected in placenta, uterus, and testis. An increase of HCG beta mRNA expression (1.7-fold) was detected at 150 micrograms/mL glycodelin-A treatment in trophoblast cell culture. Time-dependence study showed that the increase in HCG beta mRNA level was evident at 60 min after glycodelin-A treatment. In summary, we have developed a highly sensitive one-tube, one-enzyme quantitative RT-PCR system that is time-saving and avoids postamplification procedures.

Detection of contamination of vaccines with the reticuloendotheliosis virus by reverse transcriptase polymerase chain reaction (RT-PCR)

The reverse transcriptase polymerase chain reaction (RT-PCR) was applied to detect contamination of Marek's disease (MD) vaccine with eticuloendothelios s virus (REV). The env primers were used for the 1st RT-PCR to amplify the DNA fragments of REV-A and -T. The rel and env primers were used for nested-PCR to confirm the sites deleted from REV-T and REV-A. Specific amplification products were detected in the 1st RT-PCR with these primers. By nested PCR with the env and the rel primer pairs, the products originating from REV-A and -T were identified. This system, using the env primer pairs, showed a specific amplification with several REV strains (REV-T, DE, CE, KI and 0202), but no amplified product was detected with MDV, NDV, IBV or ILTV. The 1st RT-PCR detected the virus in a concentration of 10 3 in 50% fluorescent antibody infectious dosse per ml (FAID 50/ml) and the nested PCR detected 10 1 FAID 50/ml virus. The sensitivity of the RT-PCR system wasfound to be higher than that of the FA assay. This system provides a rapid, sensitive and specific method for detection of contamination of MD vaccines with REV-RNA, and it may be applied for quality control of live vaccines.

Growth of LNCaP human prostate cancer cells is stimulated by estradiol via its own receptor

We report that growth of LNCaP human prostate cancer cells is significantly stimulated (up to 120% above control) by physiological estradiol (E2) concentrations. This growth increase appears to be comparable to that induced by either testosterone or dihydrotestosterone, as also reported by others. This paper presents novel illustrative evidence for estrogen-binding proteins and messenger RNA transcripts in LNCaP cells. In fact, 1) the reverse transcriptase-polymerase chain reaction system documented normal messenger RNA for estrogen receptors (ER); 2) the radioligand binding assay allowed the detection of high affinity, reduced capacity binding sites in both soluble and nuclear cell fractions; and 3) the immunocytochemical analysis showed a consistently intensive staining for both ER and progesterone receptors. Compared to other human estrogen-responsive mammary cancer cells, MCF7 and ZR75-1, ER expression in LNCaP cells was not significantly lower, as shown by levels of the ER transcripts, number of sites per cell, or femtomoles per mg DNA as well as the percentage and intensity of immunocytochemical staining. A relative estimate of ER expression obtained by matching LNCaP with another human prostate cancer cell line, PC3, always displayed significantly and consistently higher levels in LNCaP cells. The detection of relatively high type I ER content in either cell compartment of LNCaP cells was paralleled by a highly intensive staining for progesterone receptors. In addition, evidence that the synthetic androgen R1881 did not compete for type I binding of E2 and that any E2-induced growth was completely reversed by the pure antiestrogen ICI-182,780, but unaffected by the antiandrogen Casodex, clearly suggests that the biological response of LNCaP cells to E2 is mediated via its own receptor.