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 <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 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.

scholarly journals Mammaglobin as a Novel Breast Cancer Biomarker: Multigene Reverse Transcription-PCR Assay and Sandwich ELISA

2004 ◽  
Vol 50 (11) ◽  
pp. 2069-2076 ◽  
Author(s):  
Barbara K Zehentner ◽  
David H Persing ◽  
Amadou Deme ◽  
Papa Toure ◽  
Stephen E Hawes ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Reverse Transcription ◽  
Sandwich Elisa ◽  
Clinical Staging ◽  
Diagnostic Tools ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Blood Samples ◽  
Mixed Control ◽  
Reverse Transcription Pcr

Abstract Background: The aim of this study was to examine the potential usefulness of a mammaglobin multigene reverse transcription-PCR (RT-PCR) assay and a mammaglobin sandwich ELISA as diagnostic tools in breast cancer. Methods: We studied peripheral blood samples from 147 untreated Senegalese women with biopsy-confirmed breast cancer and gathered patient information regarding demographic, and clinical staging of disease. The samples were tested for mammaglobin and three breast cancer-associated gene transcripts by a multigene real-time RT-PCR assay and for serum mammaglobin protein by a sandwich ELISA assay. Results: In 77% of the breast cancer blood samples, a positive signal was obtained in the multigene RT-PCR assay detecting mammaglobin and three complementary transcribed genes. Fifty samples from healthy female donors tested negative. Significant correlations were found between mammaglobin protein in serum, presence of mammaglobin mRNA-expressing cells in blood, stage of disease, and tumor size. Circulating mammaglobin protein was detected in 68% of the breast cancer sera, and was increased in 38% in comparison with a mixed control population. The RT-PCR assay and the ELISA for mammaglobin produced a combined sensitivity of 84% and specificity of 97%. Conclusion: The ELISA and RT-PCR for mammaglobin and mammaglobin-producing cells could be valuable tools for diagnosis and prognosis of breast cancer.

scholarly journals Time Profile of Appearance and Disappearance of Circulating Placenta-Derived mRNA in Maternal Plasma

2006 ◽  
Vol 52 (2) ◽  
pp. 313-316 ◽  
Author(s):  
Rossa WK Chiu ◽  
Wing-bong Lui ◽  
Mei-chun Cheung ◽  
Nihal Kumta ◽  
Antonio Farina ◽  
...  
Keyword(s):  
Pregnant Women ◽  
Plasma Concentrations ◽  
Maternal Blood ◽  
Maternal Plasma ◽  
Time Profile ◽  
Placental Lactogen ◽  
Human Placental Lactogen ◽  
Blood Samples ◽  
Reverse Transcription Pcr ◽  
Kinetics Of

Abstract Background: Fetal RNA of placental origin has been detected in the plasma of pregnant women, but the timing of the first appearance and the detailed kinetics of postdelivery clearance of such circulating RNA have not been studied. Methods: To address the timing of the first appearance of circulating placental RNA, we collected serial maternal blood samples from 47 women who had conceived by assisted reproductive procedures. To address the postdelivery clearance kinetics, we collected serial postdelivery blood samples from 6 pregnant women who had delivered by cesarean section. Placenta-derived transcripts were sought by real-time quantitative reverse transcription-PCR. Results: The earliest gestational age at which human placental lactogen and human chorionic gonadotropin β-subunit mRNAs were detectable in a proportion of the pregnant women was the 4th week of gestation. The postdelivery study indicated that the median apparent half-life for the clearance of human placental lactogen mRNA was 14 min. Conclusions: Placenta-derived mRNA can be found in maternal plasma from very early on in gestation, suggesting a possible role for early noninvasive prenatal diagnosis or monitoring. The rapid kinetics of circulating placental mRNA suggest that its plasma concentrations may be used to monitor recent physiologic or pathologic events.

scholarly journals Implementation of an in-house real-time reverse transcription-PCR assay to detect the emerging SARS-CoV-2 N501Y variants

2021 ◽  
pp. 104868
Author(s):  
Marielle BEDOTTO ◽  
Pierre-Edouard FOURNIER ◽  
Linda HOUHAMDI ◽  
Philippe COLSON ◽  
Didier RAOULT
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Pcr Assay ◽  
Reverse Transcription Pcr

scholarly journals Plasma γ-Globin Gene Expression Suggests that Fetal Hematopoietic Cells Contribute to the Pool of Circulating Cell-Free Fetal Nucleic Acids during Pregnancy

2004 ◽  
Vol 50 (4) ◽  
pp. 689-693 ◽  
Author(s):  
Tuangsit Wataganara ◽  
Erik S LeShane ◽  
Angela Y Chen ◽  
Lynn Borgatta ◽  
Inga Peter ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Pregnant Women ◽  
Globin Gene ◽  
Hematopoietic Cells ◽  
First Trimester ◽  
Maternal Plasma ◽  
Plasma Samples ◽  
Globin Mrna ◽  
Reverse Transcription Pcr ◽  
Gapdh Mrna

Abstract Background: Reports of placental mRNA sequences in the plasma of pregnant women suggest that the placenta is the predominant source of cell-free fetal nucleic acids in maternal plasma during pregnancy. We developed an assay for γ-globin mRNA concentrations to determine whether hematopoietic cells also contribute to the pool of fetal mRNA in maternal plasma. Methods: Frozen paired plasma samples obtained from 40 women before and within 20 min after elective first-trimester termination of pregnancy (TOP) were analyzed. Fresh plasma samples from eight nonpregnant individuals were included as controls. Plasma γ-globin mRNA was measured by use of real-time reverse transcription-PCR and analyzed with gestational age. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used to confirm the presence of cell-free RNA in each sample. Results: γ-Globin and GAPDH mRNA sequences were detected in every plasma sample. The concentrations of both messages were significantly increased in pregnancy (P <0.01). The concentrations of γ-globin mRNA were decreased in most women after TOP, but γ-globin mRNA was increased in some patients when TOP was performed later than 9 weeks of gestation. Conclusions: γ-Globin mRNA sequences can be detected and measured in fresh and frozen plasma samples. Plasma γ-globin and GAPDH mRNA concentrations are affected by pregnancy. The increased posttermination γ-globin mRNA concentrations seen in some patients suggest that the source of this message is fetal hematopoietic cells. Further study in pregnant women after 9 weeks of gestation is necessary to evaluate the potential of γ-globin mRNA as a marker for fetomaternal hemorrhage.

Comet and cup genes in Drosophila spermatogenesis: the first demonstration of post-meiotic transcription

2008 ◽  
Vol 36 (3) ◽  
pp. 540-542 ◽  
Author(s):  
Carine Barreau ◽  
Elizabeth Benson ◽  
Helen White-Cooper
Keyword(s):  
In Situ Hybridization ◽  
Expression Pattern ◽  
Reverse Transcription ◽  
Protein Product ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Single Cyst ◽  
Reverse Transcription Pcr

Post-meiotic transcription is widespread in mammalian spermatogenesis, but is generally believed to be absent from Drosophila spermatogenesis. Genes required during meiosis, in early spermatids or later in spermiogenesis are typically transcribed in primary spermatocytes in Drosophila. Their mRNAs are then stored in the cytoplasm until the protein product is needed. Recently, using in situ hybridization, we identified 17 Drosophila genes, collectively named ‘comets’ and ‘cups’, whose mRNAs are most abundant in, and localize to the distal ends of, elongating spermatids. Using a single-cyst quantitative RT–PCR (reverse transcription–PCR) assay, we confirmed this unusual expression pattern and conclusively demonstrate the existence of post-meiotic transcription in Drosophila spermatids. We found that transcription of comets and cups occurs just before protamines can be detected in spermatid nuclei.

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