scholarly journals Quantification of Melanoma Cell-specific MART-1 mRNA in Peripheral Blood by a Calibrated Competitive Reverse Transcription-PCR

2000 ◽  
Vol 46 (12) ◽  
pp. 1923-1928 ◽  
Author(s):  
Boe Sandahl Sørensen ◽  
Henrik Schmidt ◽  
Hans von der Maase ◽  
Per Thor Straten ◽  
Ebba Nexø
Keyword(s):  
Malignant Melanoma ◽  
Melanoma Cell ◽  
Reverse Transcription ◽  
Peripheral Blood ◽  
Pcr Amplification ◽  
Melanoma Cells ◽  
Rt Pcr ◽  
Blood Samples ◽  
Reverse Transcription Pcr

Abstract Background: Reverse transcription-PCR (RT-PCR) amplification of melanoma cell-specific mRNA can detect melanoma cells in the peripheral blood of patients with malignant melanoma. We present a method to quantify mRNA coding for the melanoma-specific melanoma antigen recognized by T cells #1 (MART-1) in RNA isolated from peripheral blood. Methods: To establish a calibration curve, we measured the concentration of MART-1 mRNA in SK-MEL-28 melanoma cells grown in vitro by competitive RT-PCR. Serial dilutions of these cells were used as calibrators in the assay. The assay was conducted by adding a fixed amount of a RNA internal standard to RNA isolated from either peripheral blood or the calibrators before RT-PCR amplification with MART-1 primers in a nested PCR design. The amount of MART-1 mRNA in blood samples was calculated from the calibration curve. Results: Addition of melanoma cells grown in vitro to blood from healthy donors demonstrated that the method can detect a single SK-MEL-28 melanoma cell in 1 mL of blood (1.5 × 10−21 mol MART-1 mRNA/mL). MART-1 mRNA was observed in 4 of 12 blood samples from patients with malignant melanoma, at concentrations of 3–18 × 10−21 mol MART-1 mRNA/mL of blood. No MART-1 mRNA was detected in blood samples from 25 controls without malignant melanoma. Intra- and interassay CVs were 15% (n = 12; mean = 44 × 10−21 mol MART-1 mRNA/mL) and 33% (15 samples analyzed in two different analytical runs; mean = 30 × 10−21 mol MART-1 mRNA/mL), respectively. Conclusions: Our method is the first competitive RT-PCR assay for quantification of melanoma cells in blood samples that compensates for the variation of both the reverse transcription and PCR reactions. The method allows the inclusion of control samples for continuous quality assessment.

Detection of circulating neoplastic cells by reverse-transcriptase polymerase chain reaction in malignant melanoma: association with clinical stage and prognosis.

1996 ◽  
Vol 14 (7) ◽  
pp. 2091-2097 ◽  
Author(s):  
B Mellado ◽  
D Colomer ◽  
T Castel ◽  
M Muñoz ◽  
E Carballo ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Malignant Melanoma ◽  
Prognostic Factor ◽  
Peripheral Blood ◽  
Clinical Stage ◽  
Melanoma Cells ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Circulating Neoplastic Cells

PURPOSE Circulating melanoma cells can be detected in peripheral blood by means of tyrosinase mRNA amplification by reverse-transcriptase polymerase chain reaction (RT-PCR). We conducted a prospective study to evaluate the clinical significance of the presence of circulating neoplastic cells in the blood of patients with malignant melanoma (MM). METHODS A sensitive RT-PCR assay was used to detect tyrosinase mRNA in the peripheral blood of patients with stages I to IV melanoma. Healthy subjects or patients with other malignancies were used as negative controls. RESULTS Ninety-one assessable patients were included in the study. There was a statistically significant association between RT-PCR positivity and clinical stage. Circulating melanoma cells were detected in 36% of patients with localized disease (stages I and II), in 45% of patients with regional nodal involvement (stage III), and in 94% of patients with metastatic disease (stage IV) (P < .001). In stage II-III patients who were RT-PCR-positive for mRNA tyrosinase in blood, the recurrence rate and disease-free survival were significantly worse than patients who were RT-PCR-negative. In multivariate analysis, RT-PCR was an independent prognostic factor for recurrence in patients with nonmetastatic disease (P = .002). CONCLUSION The detection of circulating melanoma cells in peripheral blood by RT-PCR correlated with the clinical stage of patients with melanoma and was an independent prognostic factor for recurrence. Further studies are warranted to better assess the significance of this test in the evaluation of prognosis, early detection of relapse, and in monitoring the effectiveness of systemic therapy.

Detection of melanoma cells in peripheral blood by tyrosinase RT-PCR in patients with malignant melanoma

1998 ◽  
Vol 16 ◽  
pp. S103
Author(s):  
Malka Hochberg ◽  
Michal Lotem ◽  
Eitan Shiloni ◽  
Claes D. Enk
Keyword(s):  
Malignant Melanoma ◽  
Peripheral Blood ◽  
Melanoma Cells ◽  
Rt Pcr

Luteolin inhibits proliferation and induces apoptosis of human melanoma cells in vivo and in vitro by suppressing MMP-2 and MMP-9 through the PI3K/AKT pathway

2019 ◽  
Vol 10 (2) ◽  
pp. 703-712 ◽  
Author(s):  
Xin Yao ◽  
Wei Jiang ◽  
Danhong Yu ◽  
Zhaowei Yan
Keyword(s):  
Malignant Melanoma ◽  
Incidence Rate ◽  
Melanoma Cell ◽  
Melanoma Cells ◽  
Human Melanoma ◽  
Akt Pathway ◽  
Human Melanoma Cells ◽  
Cell Metastasis

Since the incidence rate of malignant melanoma is increasing annually, development of drugs against melanoma cell metastasis has become more urgent.

scholarly journals Detection and Typing of Human Pathogenic Hantaviruses by Real-Time Reverse Transcription-PCR and Pyrosequencing

2007 ◽  
Vol 53 (11) ◽  
pp. 1899-1905 ◽  
Author(s):  
Marit Kramski ◽  
Helga Meisel ◽  
Boris Klempa ◽  
Detlev H Krüger ◽  
Georg Pauli ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Hantaan Virus ◽  
Hantavirus Infection ◽  
Sequence Information ◽  
Rt Pcr ◽  
Clinical Specimens ◽  
Reverse Transcription Pcr ◽  
Pcr Assays

Abstract Background: Because the clinical course of human infections with hantaviruses can vary from subclinical to fatal, rapid and reliable detection of hantaviruses is essential. To date, the diagnosis of hantavirus infection is based mainly on serologic assays, and the detection of hantaviral RNA by the commonly used reverse transcription (RT)-PCR is difficult because of high sequence diversity of hantaviruses and low viral loads in clinical specimens. Methods: We developed 5 real-time RT-PCR assays, 3 of which are specific for the individual European hantaviruses Dobrava, Puumala, or Tula virus. Two additional assays detect the Asian species Hantaan virus together with Seoul virus and the American species Andes virus together with Sin Nombre virus. Pyrosequencing was established to provide characteristic sequence information of the amplified hantavirus for confirmation of the RT-PCR results or for a more detailed virus typing. Results: The real-time RT-PCR assays were specific for the respective hantavirus species and optimized to run on 2 different platforms, the LightCycler and the ABI 7900/7500. Each assay showed a detection limit of 10 copies of a plasmid containing the RT-PCR target region, and pyrosequencing was possible with 10 to 100 copies per reaction. With this assay, viral genome could be detected in 16 of 552 (2.5%) specimens of suspected hantavirus infections of humans and mice. Conclusions: The new assays detect, differentiate, and quantify hantaviruses in clinical specimens from humans and from their natural hosts and may be useful for in vitro studies of hantaviruses.

scholarly journals Detection and Diversity of Expressed Denitrification Genes in Estuarine Sediments after Reverse Transcription-PCR Amplification from mRNA

2002 ◽  
Vol 68 (10) ◽  
pp. 5017-5025 ◽  
Author(s):  
Balbina Nogales ◽  
Kenneth N. Timmis ◽  
David B. Nedwell ◽  
A. Mark Osborn
Keyword(s):  
Reverse Transcription ◽  
Blot Hybridization ◽  
Pcr Amplification ◽  
Southern Blot Hybridization ◽  
Estuarine Sediments ◽  
Rt Pcr ◽  
Sediment Samples ◽  
Reverse Transcription Pcr ◽  
Nitrogen Inputs ◽  
Denitrification Genes

ABSTRACT The expression of five denitrification genes coding for two nitrate reductases (narG and napA), two nitrite reductases (nirS and nirK), and nitrous oxide reductase (nosZ) was analyzed by reverse transcription (RT)-PCR of mRNA extracted from two sediment samples obtained in the River Colne estuary (United Kingdom), which receives high nitrogen inputs and for which high denitrification rates have been observed. The presence of all five genes in both sediment samples was confirmed by PCR amplification from extracted DNA prior to analysis of gene expression. Only nirS and nosZ mRNAs were detected; nirS was detected directly as an RT-PCR amplification product, and nosZ was detected following Southern blot hybridization. This indicated that active expression of at least the nirS and nosZ genes was occurring in the sediments at the time of sampling. Amplified nirS RT-PCR products were cloned and analyzed by sequencing, and they were compared with amplified nirS gene sequences from isolates obtained from the same sediments. A high diversity of nirS sequences was observed. Most of the cloned nirS sequences retrieved were specific to one site or the other, which underlines differences in the compositions of the bacterial communities involved in denitrifrification in the two sediments analyzed.

scholarly journals The Concentration of Circulating Corticotropin-releasing Hormone mRNA in Maternal Plasma Is Increased in Preeclampsia

2003 ◽  
Vol 49 (5) ◽  
pp. 727-731 ◽  
Author(s):  
Enders K O Ng ◽  
Tse N Leung ◽  
Nancy B Y Tsui ◽  
Tze K Lau ◽  
Nirmal S Panesar ◽  
...  
Keyword(s):  
Pregnant Women ◽  
Gestational Age ◽  
Reverse Transcription ◽  
Peripheral Blood ◽  
Maternal Plasma ◽  
Corticotropin Releasing Hormone ◽  
Pcr Assay ◽  
Blood Samples ◽  
Reverse Transcription Pcr ◽  
Plasma Rna

Abstract Background: Increased fetal DNA in maternal plasma/serum has been reported in pregnancies complicated by preeclampsia. We hypothesize that fetal RNA may also be increased in maternal plasma in preeclampsia. Methods: We developed a real-time quantitative reverse transcription-PCR assay to measure the concentration of the mRNA of the corticotropin-releasing hormone (CRH) locus. Peripheral blood samples were obtained from healthy pregnant women both before and 2 h after delivery. Peripheral blood samples were also obtained from women suffering from preeclampsia and controls matched for gestational age. Plasma was harvested from these samples, and RNA was extracted. Plasma RNA was subjected to analysis by the reverse transcription-PCR assay. Results: CRH mRNA was detected in the plasma of 10 healthy pregnant women in the third trimester. CRH mRNA was found to be cleared very rapidly after cesarean section, with no detectable signal by 2 h postpartum. Plasma CRH mRNA concentrations were 1070 and 102 copies/mL, respectively, in 12 preeclamptic women and 10 healthy pregnant women matched for gestational age (Mann–Whitney test, P &lt;0.001). Conclusion: Plasma CRH mRNA represents a new molecular marker for preeclampsia. Maternal plasma RNA is gender- and polymorphism-independent and may allow noninvasive gene-expression profiling of an unborn fetus.

scholarly journals P2X7 promotes metastatic spreading and triggers release of miRNA-containing exosomes and microvesicles from melanoma cells

2021 ◽  
Vol 12 (12) ◽  
Author(s):  
Anna Pegoraro ◽  
Elena De Marchi ◽  
Manuela Ferracin ◽  
Elisa Orioli ◽  
Michele Zanoni ◽  
...  
Keyword(s):  
Malignant Melanoma ◽  
Melanoma Cell ◽  
P2x7 Receptor ◽  
Metastatic Malignant Melanoma ◽  
Melanoma Cells ◽  
Vesicular Release ◽  
And Migration ◽  
Metastatic Spreading

AbstractTumor growth and metastatic spreading are heavily affected by the P2X7 receptor as well as microvesicles and exosomes release into the tumor microenvironment. P2X7 receptor stimulation is known to trigger vesicular release from immune and central nervous system cells. However, P2X7 role in microvesicles and exosomes delivery from tumor cells was never analyzed in depth. Here we show that P2X7 is overexpressed in patients affected by metastatic malignant melanoma and that its expression closely correlates with reduced overall survival. Antagonism of melanoma cell-expressed P2X7 receptor inhibited in vitro anchorage-independent growth and migration and in vivo dissemination and lung metastasis formation. P2X7 stimulation triggered the release of miRNA-containing microvesicles and exosomes from melanoma cells, profoundly altering the nature of their miRNA content, as well as their dimensions and quantity. Among the more than 200 miRNAs that we found up-or-down-modulated for each vesicular fraction tested, we identified three miRNAs, miR-495-3p, miR-376c-3p, and miR-6730-3p, that were enriched in both the exosome and microvesicle fraction in a P2X7-dependent fashion. Interestingly, upon transfection, these miRNAs promoted melanoma cell growth or migration, and their vesicular release was minimized by P2X7 antagonism. Our data unveil an exosome/microvesicle and miRNA-dependent mechanism for the pro-metastatic activity of the P2X7 receptor and highlight this receptor as a suitable prognostic biomarker and therapeutic target in malignant melanoma.

scholarly journals Development of a Reverse Transcription-PCR–DNA Enzyme Immunoassay for Detection of “Norwalk-Like” Viruses and Hepatitis A Virus in Stool and Shellfish

2001 ◽  
Vol 67 (2) ◽  
pp. 742-749 ◽  
Author(s):  
Kellogg J. Schwab ◽  
Frederick H. Neill ◽  
Françoise Le Guyader ◽  
Mary K. Estes ◽  
Robert L. Atmar
Keyword(s):  
Enzyme Immunoassay ◽  
Reverse Transcription ◽  
Environmental Samples ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Pcr Amplification ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Pcr Product ◽  
Dna Enzyme Immunoassay

ABSTRACT Outbreaks of food- and waterborne gastroenteritis are being increasingly reported throughout the world. The analysis of environmental samples by newer diagnostic techniques such as reverse transcription-PCR (RT-PCR) amplification of nucleic acid has begun to identify human enteric viruses (predominantly “Norwalk-like” viruses [NLVs]) as the cause of many of these outbreaks. To streamline NLV detection from environmental samples such as shellfish, we have developed an RT-PCR–oligoprobe amplification and detection method using several new procedures that enable confirmed RT-PCR amplification and product detection in 1 day. The new steps include replacing reverse transcriptase and Taq polymerase with rTth polymerase, a heat-stable enzyme that functions as both a reverse transcriptase and DNA polymerase, in a single-tube, single-buffer, elevated temperature reaction. An internal standard Norwalk virus (NV) RNA control is added to each RT-PCR to identify sample inhibition, and thermolabile uracil N-glycosylase is incorporated into the reaction to prevent PCR product carryover contamination. Finally, RT-PCR-generated amplicons are detected in microtiter wells using virus-specific biotinylated oligoprobes in an enzyme-linked immunosorbent assay-based format. The DNA enzyme immunoassay is based on the capture of PCR product by biotinylated probes fixed onto individual streptavidin-coated wells. Using this method, low levels of NV were detected in stool and both NLV and hepatitis A virus were detected in bivalve mollusks following bioaccumulation. The method also successfully detected NLV in oysters implicated in an outbreak of NLV gastroenteritis. This method dramatically decreases the time needed for analysis and is amenable to automation.

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