Reverse Transcription of the Ribonucleic Acid: The First Step in RT-PCR Assay

2010 ◽  
pp. 261-270 ◽  
Author(s):  
Fadia Haddad ◽  
Kenneth M. Baldwin
Keyword(s):  
Reverse Transcription ◽  
Ribonucleic Acid ◽  
Rt Pcr ◽  
Pcr Assay

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 BAALC and ERG Expression in Egyptian Patients with Acute Myeloid Leukemia, Relation to Survival and Response to Treatment

2016 ◽  
Vol 4 (2) ◽  
pp. 264-270 ◽  
Author(s):  
Aml Soliman ◽  
Asmaa Abdel Aal ◽  
Reham Afify ◽  
Noha Ibrahim
Keyword(s):  
Polymerase Chain Reaction ◽  
Acute Myeloid Leukemia ◽  
Reverse Transcription ◽  
Myeloid Leukemia ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Polymerase Chain ◽  
Age And Sex ◽  
Acute Myeloid

AIM: Aim was to detect Brain and Acute Leukemia, Cytoplasmic (BAALC) and ETS-related gene (ERG) expression in patients with acute myeloid leukemia (AML) as well as to study their biologic and prognostic impact on the disease outcome and survival.PATIENTS AND METHODS: The current study was carried out on 44 patients with denovo acute myeloid leukemia, as well as 44 age and sex matched controls. The quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) assay was performed for estimation of BAALC and ERG expression.RESULTS: The current study was carried out on 44 patients with denovo acute myeloid leukemia, as well as 44 age and sex matched controls. The quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) assay was performed for estimation of BAALC and ERG expression. BAALC was expressed in 36 (81.82%) of AML cases versus 10 (22.72%) of the control group which was highly statistically significant (P < 0.001). While ERG was positive in 39(88.64%) of cases and 8(18.18 %) of controls and that was also highly statistically significant (P < 0.001).CONCLUSION: Further researches still needed to clarify the role of BAALC and ERG in the pathogenesis of leukemia and their importance as targets for treatment of AML.

Multiplex Nested Reverse Transcription-Polymerase Chain Reaction for Respiratory Viruses in Acute Otitis Media

2003 ◽  
Vol 112 (3) ◽  
pp. 252-257 ◽  
Author(s):  
Toshio Ishibashi ◽  
Hiroko Monobe ◽  
Masanobu Shinogami ◽  
Yuka Nomura ◽  
Jun Yano
Keyword(s):  
Polymerase Chain Reaction ◽  
Otitis Media ◽  
Reverse Transcription ◽  
Acute Otitis Media ◽  
Respiratory Viruses ◽  
Molecular Technique ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Polymerase Chain

Because respiratory viruses play an important role in the causation and pathogenesis of acute otitis media (AOM), determining which virus has infected a child is important with respect to vaccines and antiviral drugs. In some instances, this information might be used to prevent the occurrence of AOM. We used a rapid, economical, and sensitive diagnostic system involving a multiplex nested reverse transcription–polymerase chain reaction (RT-PCR) assay to detect various respiratory viruses in clinical specimens of middle ear fluid (MEF) from children with AOM in our hospital. Multiplex RT-PCR was completed on 40 MEF samples from 28 infants and children less than 6 years old with AOM. Viral RNA was detected in 17 MEF samples (43%). Respiratory syncytial virus type A was present in 12 samples, adenovirus in 3, rhinovirus in 2, and influenza A (H3N2) in 1. The multiplex RT-PCR assay is recommended to clinical laboratories that are considering adoption of a molecular technique for viral diagnosis.

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.

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.

Increased detection of rotavirus using a real time reverse transcription-polymerase chain reaction (RT-PCR) assay in stool specimens from children with diarrhea

2004 ◽  
Vol 72 (3) ◽  
pp. 496-501 ◽  
Author(s):  
Xiaoli L. Pang ◽  
Bonita Lee ◽  
Nasim Boroumand ◽  
Barbara Leblanc ◽  
Jutta K. Preiksaitis ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Reverse Transcription ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Stool Specimens ◽  
Polymerase Chain

Investigations on the Frequency of Norovirus Contamination of Ready-to-Eat Food Items in Istanbul, Turkey, by Using Real-Time Reverse Transcription PCR

2011 ◽  
Vol 74 (5) ◽  
pp. 840-843 ◽  
Author(s):  
AYSUN YILMAZ ◽  
KAMIL BOSTAN ◽  
EDA ALTAN ◽  
KARLO MURATOGLU ◽  
NURI TURAN ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Epidemiological Studies ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Food Items ◽  
Reverse Transcription Pcr ◽  
Significant Difference ◽  
Tomato Sample ◽  
Pcr Assays

Investigation of norovirus (NoV) contamination of food items is important because many outbreaks occur after consumption of contaminated shellfish, vegetables, fruits, and water. The frequency of NoV contamination in food items has not previously been investigated in Turkey. The aim of this study was to investigate the frequency of human NoV genogroups (G) I and II in ready-to-eat tomatoes, parsley, green onion, lettuce, mixed salads, and cracked wheat balls. RNA was extracted with the RNeasy Mini Kit, and a real-time reverse transcription (RT) PCR assay was performed using primers specific for NoV GI and GII. Among the 525 samples analyzed, NoV GII was detected in 1 green onion sample and 1 tomato sample by both SYBR Green and TaqMan real-time RT-PCR assays; no GI virus was detected. The Enterobactericaeae and Escherichia coli levels in the NoV-positive green onion were 6.56 and 1.28 log CFU/g, and those in the tomato were 5.55 and 1.30 log CFU/g, respectively. No significant difference in the bacterial levels was found between the NoV-positive and NoV-negative samples. This study is the first in which NoV GII was found in ready-to-eat food collected from Istanbul, Turkey; thus, these foods may be considered a risk to human health. Epidemiological studies and measures to prevent NoV infection should be considered.

scholarly journals Improved Detection of Rhinoviruses in Clinical Samples by Using a Newly Developed Nested Reverse Transcription-PCR Assay

1999 ◽  
Vol 37 (3) ◽  
pp. 524-530 ◽  
Author(s):  
Arno C. Andeweg ◽  
Theo M. Bestebroer ◽  
Martijn Huybreghs ◽  
Tjeerd G. Kimman ◽  
Jan C. de Jong
Keyword(s):  
Reverse Transcription ◽  
Rna Extraction ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Noncoding Regions ◽  
Reverse Transcription Pcr ◽  
Highly Sensitive ◽  
Virus Culture ◽  
Culture Positive

This paper describes the development and evaluation of a new nested reverse transcription (RT)-PCR for the detection of rhinovirus in clinical samples. The nucleotide sequences of the 5′ noncoding regions of 39 rhinoviruses were determined in order to map the most conserved subregions. We designed a set of rhinovirus-specific primers and probes directed to these subregions and developed a new nested RT-PCR. The new assay includes an optimal RNA extraction method and amplicon identification with probe hybridization to discriminate between rhinoviruses and the closely related enteroviruses. It proved to be highly sensitive and specific. When tested on a dilution series of cultured viruses, the new PCR protocol scored positive at 10- to 100-fold-higher dilutions than a previously used nested RT-PCR. When tested on a collection of clinical samples obtained from 1,070 acute respiratory disease patients who had consulted their general practitioners, the new assay demonstrated a rhinovirus in 24% of the specimens, including all culture-positive samples, whereas the previously used PCR assay or virus culture detected a rhinovirus in only 3.5 to 6% of the samples. This new assay should help determine the disease burden associated with rhinovirus infections.

scholarly journals Impact of COVID-19 pre-test probability on positive predictive value of high cycle threshold SARS-CoV-2 real-time reverse transcription PCR test results

2021 ◽  
pp. 1-18
Author(s):  
Jonathan B. Gubbay ◽  
Heather Rilkoff ◽  
Heather L. Kristjanson ◽  
Jessica D. Forbes ◽  
Michelle Murti ◽  
...  
Keyword(s):  
Positive Predictive Value ◽  
Real Time ◽  
Reverse Transcription ◽  
Predictive Value ◽  
Large Scale ◽  
Rdrp Gene ◽  
Nucleic Acid Detection ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Test Probability

Abstract Objectives Performance characteristics of SARS-CoV-2 nucleic acid detection assays are understudied within contexts of low pre-test probability, including screening asymptomatic persons without epidemiological links to confirmed cases, or asymptomatic surveillance testing. SARS-CoV-2 detection without symptoms may represent presymptomatic or asymptomatic infection, resolved infection with persistent RNA shedding, or a false positive test. This study assessed positive predictive value of SARS-CoV-2 real-time reverse transcription polymerase chain reaction (rRT-PCR) assays by retesting positive specimens from five pre-test probability groups ranging from high to low with an alternate assay. Methods A total of 122 rRT-PCR positive specimens collected from unique patients between March and July 2020 were retested using a laboratory-developed nested RT-PCR assay targeting the RNA-dependent RNA polymerase (RdRp) gene followed by Sanger sequencing. Results Significantly fewer (15.6%) positive results in the lowest pre-test probability group (facilities with institution-wide screening having ≤ 3 positive asymptomatic cases) were reproduced with the nested RdRp gene RT-PCR assay than in each of the four groups with higher pre-test probability (individual group range 50·0% to 85·0%). Conclusions Large-scale SARS-CoV-2 screening testing initiatives among low pre-test probability populations should be evaluated thoroughly prior to implementation given the risk of false positives and consequent potential for harm at the individual and population level.

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