Total RNA‐Isolation of Abdominal Hernia of Rats for Quantitative Real‐Time Reverse Transcription (RT) PCR Assays

We describe automated nucleic acid (NA) isolation from diverse sample types using MagMAX kits (Ambion, Inc.) on KingFisher Magnetic Particle Processors (Thermo Scientific). The MagMAX-96 Blood RNA Isolation Kit is designed for total RNA isolation from whole blood from several species, without white blood cell fractionation, in about 45 min (including genomic DNA removal). The MagMAX-96 Total RNA Isolation Kit is designed for total RNA isolation from up to 2 × 10 6 cultured cells and up to 10-mg tissue. The isolated total RNA is highly intact and pure, ready to use in downstream applications such as microarray analysis or real-time reverse transcription (RT)-PCR for gene expression profiling or pathogen detection. The MagMAX-96 Viral RNA Isolation Kit is designed for viral RNA and DNA isolation from cell free or nearly cell-free samples such as swabs, serum, and plasma; it takes about 15 min. Total NA of high quality and purity is recovered at >75% efficiency, providing high sensitivity for pathogen detection by real-time RT-PCR. Unlike automated liquid handling systems that move reagents into and out of a single well of a multiwell plate to perform the different steps of an RNA isolation procedure, the KingFisher Magnetic Particle Processors use permanent magnetic rods to collect magnetic beads from solution and release them into another well containing reagent for the subsequent step of the procedure. The effectiveness of bead collection and transfer lead to superior washing and elution efficiency, as well as rapid processing. It is a very effective strategy for automation of magnetic-bead-based NA isolation kits. (JALA 2007; 12:195–201)

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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

Clinical Chemistry ◽

10.1373/clinchem.2008.117952 ◽

2009 ◽

Vol 55 (4) ◽

pp. 765-773 ◽

Cited By ~ 93

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.

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Performance of Single-Step Gel-Based Reverse Transcription-PCR (RT-PCR) Assays Equivalent to That of Real-Time RT-PCR Assays for Detection of the Severe Acute Respiratory Syndrome-Associated Coronavirus

Journal of Clinical Microbiology ◽

10.1128/jcm.43.8.4262-4265.2005 ◽

2005 ◽

Vol 43 (8) ◽

pp. 4262-4265 ◽

Cited By ~ 1

Author(s):

M. Inoue ◽

T. Barkham ◽

L. K. Keong ◽

L. S. Gee ◽

H. Wanjin

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Real Time ◽

Reverse Transcription ◽

Single Step ◽

Rt Pcr ◽

Reverse Transcription Pcr ◽

Pcr Assays

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

Journal of Food Protection ◽

10.4315/0362-028x.jfp-10-475 ◽

2011 ◽

Vol 74 (5) ◽

pp. 840-843 ◽

Cited By ~ 9

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.

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

Clinical Chemistry ◽

10.1373/clinchem.2007.093245 ◽

2007 ◽

Vol 53 (11) ◽

pp. 1899-1905 ◽

Cited By ~ 64

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.

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Molecular (real-time reverse transcription polymerase chain reaction) diagnosis of SARS-CoV-2 infections: complexity and challenges

Journal of Laboratory Medicine ◽

10.1515/labmed-2020-0135 ◽

2021 ◽

Vol 45 (3) ◽

pp. 135-142

Author(s):

Shneh Sethi ◽

Trinad Chakraborty

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Reverse Transcription ◽

Fatal Outcome ◽

Respiratory Illness ◽

World Health ◽

Rt Pcr ◽

Chain Reaction ◽

Polymerase Chain ◽

Pcr Assays

Abstract The outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first recorded in Wuhan, China. The World Health Organization initially classified COVID-19 as a public health emergency and subsequently declared the disease a global pandemic. COVID-19 can take at least three distinct forms: severe acute distress syndrome with a potentially fatal outcome, mild respiratory illness (pneumonia with eventual recovery) and asymptomatic infection. All three disease forms have the potential to transmit the infection to healthy contacts. At present, real-time reverse transcription polymerase chain reaction (RT-PCR) is the only available laboratory tool to confirm the presence of viral RNA in patient specimens. These assays are designed to detect one or more (at least 2) SARS-CoV-2 RNA gene targets allowing the detection of the virus. Commercially available RT-PCR assays employ various gene targets of the viral genome in their assay systems. Additionally, there are differences in primer selection for the same gene region of SARS-CoV-2. At present, it is unclear whether the results from different RT-PCR assays are comparable in detecting the spectrum of COVID-19 manifestations. The purpose of the present article is twofold: first, to briefly focus on the findings of these reports; and second, to emphasize the various challenges and flaws that can potentially impact the diagnostic accuracy of RT-PCR testing for SARS-CoV-2.

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Rapid Quantification of Human ABCA1 mRNA in Various Cell Types and Tissues by Real-Time Reverse Transcription-PCR

Clinical Chemistry ◽

10.1093/clinchem/47.12.2089 ◽

2001 ◽

Vol 47 (12) ◽

pp. 2089-2097 ◽

Cited By ~ 67

Author(s):

Danuta Kielar ◽

Wolfgang Dietmaier ◽

Thomas Langmann ◽

Charalampos Aslanidis ◽

Mario Probst ◽

...

Keyword(s):

Real Time ◽

Internal Control ◽

Reverse Transcription ◽

Calibration Curve ◽

Cultured Cells ◽

Rt Pcr ◽

Total Rna ◽

Reverse Transcription Pcr ◽

Detection And Quantification ◽

Abca1 Mrna

Abstract Background: The ABCA1 gene encodes for a member of subfamily A of the ATP-binding cassette transporters that plays an important role in cellular export of cholesterol and phospholipids; therefore, quantification of the ABCA1 mRNA is critical in many studies related to its expression and regulation by metabolic factors, nutritional status, and new antiatherogenic drug candidates. We developed a rapid, sensitive, specific, and reproducible real-time reverse transcription-PCR (RT-PCR) method for detection and quantification of ABCA1 transcripts in total RNA isolated from cultured human cells and tissues. Methods: To quantify ABCA1 mRNA, we generated a calibration curve from serial dilutions of in vitro-transcribed RNA corresponding to an amplified ABCA1 cDNA 205-bp fragment (homologous calibrator). Two pairs of fluorescent hybridization probes were used to detect the ABCA1 and porphobilinogen deaminase (PBGD) mRNAs; the latter served as an internal control. PCR was performed as real-time amplification of ABCA1 mRNA in 100 ng of total RNA isolated from various human tissues, and cultured cells were calculated from the calibration curve. In addition, normalized values of target (ABCA1/PBGD ratio) were calculated. Results: Using this method, we quantified ABCA1 transcripts in various human tissue samples as well as in monocytes, THP-1 cells, fibroblasts, and adipocytes. We demonstrated ABCA1 mRNA up-regulation during human adipocyte and monocyte differentiation. In addition, we examined the effect of cholesterol loading and deloading on ABCA1 expression in monocytes, THP-1 cells, and fibroblasts. Conclusions: Our RT-PCR assay allows the specific and highly reproducible detection and quantification of minute amounts of human ABCA1 mRNA. This new method is more accurate, more informative, and less laborious than the classic RT-PCR methods and Northern blot; it therefore could simplify all studies on ABCA1 mRNA expression.

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Monitoring Shedding of Five Genotypes of RotaTeq Vaccine Viruses by Genotype-Specific Real-Time Reverse Transcription-PCR Assays

Journal of Clinical Microbiology ◽

10.1128/jcm.00035-18 ◽

2018 ◽

Vol 56 (6) ◽

Cited By ~ 4

Author(s):

Yuki Higashimoto ◽

Masaru Ihira ◽

Yu Miyazaki ◽

Ayumi Kuboshiki ◽

Sayaka Yoshinaga ◽

...

Keyword(s):

Real Time ◽

Reverse Transcription ◽

Cross Reactivity ◽

Rt Pcr ◽

Fecal Shedding ◽

Reverse Transcription Pcr ◽

Coefficients Of Variation ◽

Long Time ◽

Stool Samples ◽

Pcr Assays

ABSTRACTRotaTeq (RV5) is a widely used live attenuated pentavalent rotavirus (RV) vaccine. Although fecal shedding of RV vaccine strains persists for long time periods, it is unclear how each vaccine strain replicates in intestinal tissue and is excreted in stool. To examine this issue, we established RV5 genotype-specific real-time reverse transcription-PCR (RT-PCR) assays. Five real-time RT-PCR assays were designed for the VP7 gene in genotypes G1, G2, G3, G4, and G6. All assays exhibited excellent linearity, and the detection limit was 1 infectious unit (IU)/reaction for G2, G4, and G6 and 10 IUs/reaction for G1 and G3. No cross-reactivity was observed among G genotypes. The inter- and intra-assay coefficients of variation were less than 3%. The assays were used to examine 129 stool samples collected from eight infants who received RV5. In cases 1 and 2, who received three rounds of vaccination, RV shedding decreased gradually with the number of vaccinations. G1 and G6 shedding appeared to be predominant in comparison to shedding of the other genotypes. Patterns of fecal shedding of the five genotypes of vaccine viruses differed between the eight vaccine recipients. RV5 genotype-specific real-time RT-PCR assays will be useful to study the molecular biology of RV5 replication in infants and experimental animals.

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