scholarly journals Quantitative Reverse Transcription-PCR Assay with an Internal Standard for the Detection of Prostate-specific Antigen mRNA

1999 ◽  
Vol 45 (9) ◽  
pp. 1397-1407 ◽  
Author(s):  
Alice Ylikoski ◽  
Minna Sjöroos ◽  
Åke Lundwall ◽  
Matti Karp ◽  
Timo Lövgren ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Detection Limit ◽  
Prostate Specific Antigen ◽  
Reverse Transcription ◽  
Specific Antigen ◽  
Internal Standard ◽  
Quantitative Detection ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Reverse Transcription Pcr

Abstract Background: Circulating prostate cells can be detected with a reverse transcription-PCR (RT-PCR) assay for prostate-specific antigen (PSA) mRNA. We have developed a new quantitative RT-PCR method for measuring PSA mRNA. Methods: The method uses a PSA-like internal standard (IS) mRNA that is added into the sample at the beginning of the RNA extraction and coamplified by RT-PCR with the PSA in the sample. After PCR amplification, the IS and PSA products are selectively detected by hybridization in a microtitration plate using probes labeled with fluorescent europium chelates. Results: The method was validated with PSA and IS mRNAs and PSA-expressing cells to obtain a detection limit of 50 PSA mRNA copies (i.e., signal 2 times the mean of zero signal), linearity up to 106 copies, and detection of a single PSA-expressing cell. In preliminary evaluations, 60% (n = 10) of the prostate cancer patients with skeletal metastases gave results above the detection limit (500 PSA mRNA copies in 5 mL of blood). The total number of PSA copies ranged from 900 ± 200 to 44 100 ± 4900 (mean ± SD) in the samples, corresponding to ∼1–100 PSA-expressing cells in 5 mL of blood. In the controls (n = 34), none of the healthy females and 2 of 19 healthy males had detectable PSA mRNA [700 ± 100 and 2000 ± 900 (mean ± SD) PSA mRNA copies in 5 mL of blood for the 2 males]. Conclusions: The assay provides sensitive and quantitative detection of PSA mRNA expression from blood samples and can be used to establish the clinically significant number of PSA mRNA copies in prostate cancer.

scholarly journals Circulating Prostate Tumor Cells Detected by Reverse Transcription-PCR in Men with Localized or Castration-Refractory Prostate Cancer: Concordance with CellSearch Assay and Association with Bone Metastases and with Survival

2009 ◽  
Vol 55 (4) ◽  
pp. 765-773 ◽  
Author(s):  
Pauliina Helo ◽  
Angel M Cronin ◽  
Daniel C Danila ◽  
Sven Wenske ◽  
Rita Gonzalez-Espinoza ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Bone Metastases ◽  
Healthy Volunteers ◽  
Real Time ◽  
Tumor Cells ◽  
Reverse Transcription ◽  
Specific Antigen ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Pcr Assays

Abstract Background: Reverse transcription-PCR (RT-PCR) assays have been used for analysis of circulating tumor cells (CTCs), but their clinical value has yet to be established. We assessed men with localized prostate cancer or castration-refractory prostate cancer (CRPC) for CTCs via real-time RT-PCR assays for KLK3 [kallikrein-related peptidase 3; i.e., prostate-specific antigen (PSA)] and KLK2 mRNAs. We also assessed the association of CTCs with disease characteristics and survival. Methods: KLK3, KLK2, and PSCA (prostate stem cell antigen) mRNAs were measured by standardized, quantitative real-time RT-PCR assays in blood samples from 180 localized-disease patients, 76 metastatic CRPC patients, and 19 healthy volunteers. CRPC samples were also tested for CTCs by an immunomagnetic separation system (CellSearch™; Veridex) approved for clinical use. Results: All healthy volunteers were negative for KLK mRNAs. Results of tests for KLK3 or KLK2 mRNAs were positive (≥80 mRNAs/mL blood) in 37 patients (49%) with CRPC but in only 15 patients (8%) with localized cancer. RT-PCR and CellSearch CTC results were strongly concordant (80%–85%) and correlated (Kendall τ, 0.60–0.68). Among CRPC patients, KLK mRNAs and CellSearch CTCs were closely associated with clinical evidence of bone metastases and with survival but were only modestly correlated with serum PSA concentrations. PSCA mRNA was detected in only 7 CRPC patients (10%) and was associated with a positive KLK mRNA status. Conclusions: Real-time RT-PCR assays of KLK mRNAs are highly concordant with CellSearch CTC results in patients with CRPC. KLK2/3-expressing CTCs are common in men with CRPC and bone metastases but are rare in patients with metastases diagnosed only in soft tissues and patients with localized cancer.

scholarly journals Numeric Definition of the Clinical Performance of the Nested Reverse Transcription-PCR for Detection of Hematogenous Epithelial Cells and Correction for Specific mRNA of Non-Target Cell Origin as Evaluated for Prostate Cancer Cells

2003 ◽  
Vol 49 (9) ◽  
pp. 1458-1466 ◽  
Author(s):  
Denis Schamhart ◽  
Johannes Swinnen ◽  
Karl-Heinz Kurth ◽  
Alex Westerhof ◽  
Ron Kusters ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Reverse Transcription ◽  
Clinical Performance ◽  
Specific Antigen ◽  
Rt Pcr ◽  
Frequency Distributions ◽  
Reverse Transcription Pcr ◽  
The Mean ◽  
Pcr Assays ◽  
Definition Of

Abstract Background: Inappropriate quality management of reverse transcription-PCR (RT-PCR) assays for the detection of blood-borne prostate cancer (PCa) cells hampers clinical conclusions. Improvement of the RT-PCR methodology for prostate-specific antigen (PSA) mRNA should focus on an appropriate numeric definition of the performance of the assay and correction for PSA mRNA that is not associated with PCa cells. Methods and Results: Repeated (RT-)PCR tests for PSA mRNA in single blood specimens from PCa patients and PCa-free controls, performed by four international institutions, showed a large percentage (≈50%) of divergent test results. The best estimates of the mean, λ (SD), of the expected Poisson frequency distributions of the number of positive tests among five replicate assays of samples from PCa-free individuals were 1.0 (0.2) for 2 × 35 PCR cycles and 0.2 (0.1) for 2 × 25 PCR cycles. Assessment of the numeric value of the mean can be considered as a new indicator of the performance of a RT-PCR assay for PSA mRNA under clinical conditions. Moreover, it determines the required number of positive test repetitions to differentiate between true and false positives for circulating prostate cells. At a predefined diagnostic specificity of ≥98%, repeated PCRs with λ of either 1.0 or 0.2 require, respectively, more than three or more than one positive tests to support the conclusion that PSA mRNA-containing cells are present. Conclusions: Repeated nested PCR tests for PSA and appropriate handling of the data allow numeric quantification of the performance of the assay and differentiation between analytical false and true positives at a predefined accuracy. This new approach may contribute to introduction of PSA RT-PCR assays in clinical practice.

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.

scholarly journals Quantification of Hepatitis A Virus in Shellfish by Competitive Reverse Transcription-PCR with Coextraction of Standard RNA

1999 ◽  
Vol 65 (1) ◽  
pp. 322-326 ◽  
Author(s):  
Charlotte Arnal ◽  
Virginie Ferre-Aubineau ◽  
Berangere Mignotte ◽  
Berthe Marie Imbert-Marcille ◽  
Sylviane Billaudel
Keyword(s):  
Tissue Culture ◽  
Reverse Transcription ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Internal Standard ◽  
Wild Type ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Reproducible Method ◽  
Dna Enzyme Immunoassay

ABSTRACT To quantify hepatitis A virus (HAV) in experimentally contaminated mussels, we developed an internal standard RNA with a 7-nucleotide deletion for competitive reverse transcription (RT)-PCR. Deposited directly into the sample, this standard was used both as extraction control and as quantification tool. After coextraction and competitive RT-PCR, standard and wild-type products were detected by differential hybridization with specific probes and a DNA enzyme immunoassay. The quantifiable range with this reproducible method was 104 to 107 copies of HAV/gram or 400 to 106 50% tissue culture infective doses/ml.

scholarly journals Evaluation of the PAX8/PPARG Translocation in Follicular Thyroid Cancer with a 4-Color Reverse-Transcription PCR Assay and Automated High-Resolution Fragment Analysis

2010 ◽  
Vol 56 (3) ◽  
pp. 391-398 ◽  
Author(s):  
Alicia Algeciras-Schimnich ◽  
Dragana Milosevic ◽  
Bryan McIver ◽  
Heather Flynn ◽  
Honey V Reddi ◽  
...  
Keyword(s):  
Thyroid Cancer ◽  
High Resolution ◽  
Reverse Transcription ◽  
Thyroid Tissue ◽  
Molecular Testing ◽  
Analytical Sensitivity ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Fragment Analysis ◽  
Reverse Transcription Pcr

Abstract Background: Molecular testing of thyroid malignancies, in combination with cytologic and histologic examination, is becoming increasingly attractive as a tool for refining traditional morphologic diagnosis. The molecular changes associated with follicular thyroid carcinoma (FTC) are point mutations in RAS oncogenes or the presence of PAX8/PPARG (paired box 8/peroxisome proliferator-activated receptor gamma) rearrangement. Methods: We developed and validated a clinical assay for the detection of PAX8/PPARG rearrangements that uses a 4-color reverse-transcription PCR (RT-PCR) assay and high-resolution fragment analysis. Results: The RT-PCR assay is applicable for detecting the various described fusion transcripts of PAX8/PPARG in formalin-fixed, paraffin-embedded thyroid tissue and in fine-needle aspirate biopsy washes from thyroid nodules. The analytical sensitivity of the assay is 1 abnormal cell in a background of 100–10 000 translocation-negative cells. A comparison of the RT-PCR assay with dual-fusion fluorescence in situ hybridization showed an overall concordance of 95%. With this assay, we obtained a prevalence for the PAX8/PPARG rearrangement in FTC of 62% (13 of 21 cases), compared with a 5% prevalence (3 of 55) for other follicular cell–derived neoplasms. Conclusions: The introduction of this assay into clinical practice could provide useful information for the diagnosis and possibly for the prognosis and treatment of thyroid cancer in the future.

VALIDATION OF QUANTITATIVE RT-PCR ASSAY FOR DETECTION OF PROSTATE SPECIFIC ANTIGEN (PSA) mRNA. MULTICENTER STUDY

1999 ◽  
pp. 239
Author(s):  
Alice Ylikoski ◽  
Timo Lovgren ◽  
Jan Trapman ◽  
Freddy Hamdy ◽  
Jack Schalken ◽  
...  
Keyword(s):  
Prostate Specific Antigen ◽  
Multicenter Study ◽  
Specific Antigen ◽  
Rt Pcr ◽  
Pcr Assay

scholarly journals Rapid and Quantitative Detection of Hepatitis A Virus from Green Onion and Strawberry Rinses by Use of Real-Time Reverse Transcription-PCR

2005 ◽  
Vol 71 (9) ◽  
pp. 5624-5626 ◽  
Author(s):  
X. C. Shan ◽  
P. Wolffs ◽  
M. W. Griffiths
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Quantitative Detection ◽  
Pcr Assay ◽  
Reverse Transcription Pcr ◽  
Immunomagnetic Capture ◽  
Capture Method

ABSTRACT In this study, an immunomagnetic capture method and a real-time reverse transcription-PCR assay were used to quantify hepatitis A virus (HAV) in green onion and strawberry rinses. This combined protocol detected as low as 0.5 PFU HAV in produce rinses and concentrated HAV levels up to 20-fold.

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