scholarly journals Nuclear Run-On Assay Using Biotin Labeling, Magnetic Bead Capture and Analysis by Fluorescence-Based RT-PCR

BioTechniques ◽  
2000 ◽  
Vol 29 (5) ◽  
pp. 1012-1017 ◽  
Author(s):  
G. Patrone ◽  
F. Puppo ◽  
R. Cusano ◽  
M. Scaranari ◽  
I. Ceccherini ◽  
...  
Keyword(s):  
Magnetic Bead ◽  
Rt Pcr ◽  
Biotin Labeling

Magnetic bead RT-PCR: establishment of a new method for detecting circulating melanoma cells

2002 ◽  
Vol 12 (4) ◽  
pp. 309-317 ◽  
Author(s):  
J. Georgieva ◽  
A. Milling ◽  
C. E. Orfanos ◽  
C. C. Geilen
Keyword(s):  
Magnetic Bead ◽  
Melanoma Cells ◽  
New Method ◽  
Rt Pcr

scholarly journals Comparison of RNA extraction methods for the detection of SARS-CoV-2 by RT-PCR

2020 ◽  
Author(s):  
Ossia M Eichhoff ◽  
Elisa Bellini ◽  
Reto Lienhard ◽  
Wendelin J. Stark ◽  
Philippe Bechtold ◽  
...  
Keyword(s):  
Magnetic Beads ◽  
Diagnostic Testing ◽  
Rna Extraction ◽  
Magnetic Bead ◽  
Extraction Methods ◽  
Rt Pcr ◽  
Diagnostic Quality ◽  
Viral Spread ◽  
Significant Difference ◽  
Rna Extraction Methods

Objectives: The SARS-CoV-2 pandemic outbreak has stressed health care systems as well as medical supply chains, but diagnostic testing is an essential public health measure to control viral spread. Here we test the suitability of different RNA extraction methods for integration into a diagnostic workflow for coronavirus testing. Methods: We applied six RNA extraction methods on the same 24 SARS-CoV-2 positive patient samples and quantified their results by subsequent reverse-transcriptase PCR (RT-PCR) of three viral genes. These methods included a) column-based extraction, b) phenol-chloroform extraction, as well as c) extraction using magnetic beads (i.e., one commercial kit as well as three different magnetic beads in combination with home-brewed buffers and solutions). Results: We achieved diagnostic-quality RT-PCR results with all methods, and there was no significant difference between the tested methods, except for one magnetic bead protocol with home-brewed buffers, in which the number of positive tested genes was significantly lower. Conclusions: Five of the six RNA extraction methods are interchangeable in a diagnostic workflow. Since some methods are more scalable than others, and have comparable results on RT-PCR quantitation, they may be more amenable to high-throughput sample processing pipelines.

scholarly journals High-Volume Extraction of Nucleic Acids by Magnetic Bead Technology for Ultrasensitive Detection of Bacteria in Blood Components

2007 ◽  
Vol 53 (1) ◽  
pp. 104-110 ◽  
Author(s):  
Melanie Störmer ◽  
Knut Kleesiek ◽  
Jens Dreier
Keyword(s):  
Detection Limit ◽  
Nucleic Acid ◽  
Nucleic Acids ◽  
Real Time ◽  
High Volume ◽  
Magnetic Bead ◽  
Locked Nucleic Acid ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Probe System

Abstract Background: Nucleic acid isolation, the most technically demanding and laborious procedure performed in molecular diagnostics, harbors the potential for improvements in automation. A recent development is the use of magnetic beads covered with nucleic acid–binding matrices. We adapted this technology with a broad-range 23S rRNA real-time reverse transcription (RT)-PCR assay for fast and sensitive detection of bacterial contamination of blood products. Methods: We investigated different protocols for an automated high-volume extraction method based on magnetic-separation technology for the extraction of bacterial nucleic acids from platelet concentrates (PCs). We added 2 model bacteria, Staphylococcus epidermidis and Escherichia coli, to a single pool of apheresis-derived, single-donor platelets and assayed the PCs by real-time RT-PCR analysis with an improved primer–probe system and locked nucleic acid technology. Coamplification of human β2-microglobulin mRNA served as an internal control (IC). We used probit analysis to calculate the minimum concentration of bacteria that would be detected with 95% confidence. Results: For automated magnetic bead–based extraction technology with the real-time RT-PCR, the 95% detection limit was 29 × 103 colony-forming units (CFU)/L for S. epidermidis and 22 × 103 CFU/L for E. coli. No false-positive results occurred, either due to nucleic acid contamination of reagents or externally during testing of 1030 PCs. Conclusions: High-volume nucleic acid extraction improved the detection limit of the assay. The improvement of the primer–probe system and the integration of an IC make the RT-PCR assay appropriate for bacteria screening of platelets.

Comparison of three magnetic-bead-based RNA extraction methods for detection of cucumber green mottle mosaic virus by real-time RT-PCR

2015 ◽  
Vol 160 (7) ◽  
pp. 1791-1796 ◽  
Author(s):  
Xiaoli Zhao ◽  
Qi Zhou ◽  
Lijie Zhang ◽  
Wenlong Yan ◽  
Ning Sun ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Real Time ◽  
Rna Extraction ◽  
Magnetic Bead ◽  
Extraction Methods ◽  
Rt Pcr ◽  
Rna Extraction Methods

Detection of Nucleic Acids in Cells or Tissue Sections Using In situ Polymerase Chain Reaction (PCR)

1996 ◽  
Vol 54 ◽  
pp. 16-17
Author(s):  
J. R. Hully ◽  
K. R. Luehrsen ◽  
K. Aoyagi ◽  
C. Shoemaker ◽  
R. Abramson
Keyword(s):  
Nucleic Acids ◽  
Viral Infections ◽  
Anatomical Location ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Cellular Source ◽  
Gene Transcripts ◽  
Initial Description ◽  
In Cells

The development of PCR technology has greatly accelerated medical research at the genetic and molecular levels. Until recently, the inherent sensitivity of this technique has been limited to isolated preparations of nucleic acids which lack or at best have limited morphological information. With the obvious exception of cell lines, traditional PCR or reverse transcription-PCR (RT-PCR) cannot identify the cellular source of the amplified product. In contrast, in situ hybridization (ISH) by definition, defines the anatomical location of a gene and/or it’s product. However, this technique lacks the sensitivity of PCR and cannot routinely detect less than 10 to 20 copies per cell. Consequently, the localization of rare transcripts, latent viral infections, foreign or altered genes cannot be identified by this technique. In situ PCR or in situ RT-PCR is a combination of the two techniques, exploiting the sensitivity of PCR and the anatomical definition provided by ISH. Since it’s initial description considerable advances have been made in the application of in situ PCR, improvements in protocols, and the development of hardware dedicated to in situ PCR using conventional microscope slides. Our understanding of the importance of viral latency or viral burden in regards to HIV, HPV, and KSHV infections has benefited from this technique, enabling detection of single viral copies in cells or tissue otherwise thought to be normal. Clearly, this technique will be useful tool in pathobiology especially carcinogenesis, gene therapy and manipulations, the study of rare gene transcripts, and forensics.

1089: Prognostic Value of Molecular Staging of Bladder Cancer with RT -PCR Assay for KRT20: Results at 5 Year-Follow-up

2007 ◽  
Vol 177 (4S) ◽  
pp. 360-360
Author(s):  
Ana Agud ◽  
Maria J. Ribal ◽  
Lourdes Mengual ◽  
Mercedes Marin-Aguilera ◽  
Laura Izquierdo ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Prognostic Value ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Molecular Staging

531: Quantitative Real-Time RT-PCR for AMACR Distinguishes Prostate Cancer with High Specificity From Benign Lesions

2005 ◽  
Vol 173 (4S) ◽  
pp. 145-145 ◽  
Author(s):  
Martin Schostak ◽  
Hans Krause ◽  
Jens Köllermann ◽  
Mark Schrader ◽  
Bernd Straub ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Real Time ◽  
High Specificity ◽  
Rt Pcr ◽  
Benign Lesions

1504: Molecular Diagnosis and Staging of Prostate Cancer Using Clusterin in Real Time Reverse Transcription Polymerase Chain Reaction (RT-PCR)

2006 ◽  
Vol 175 (4S) ◽  
pp. 485-486
Author(s):  
Sabarinath B. Nair ◽  
Christodoulos Pipinikas ◽  
Roger Kirby ◽  
Nick Carter ◽  
Christiane Fenske
Keyword(s):  
Prostate Cancer ◽  
Polymerase Chain Reaction ◽  
Real Time ◽  
Molecular Diagnosis ◽  
Reverse Transcription ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain

1650: Novel Gene Expression Markers in Renal Tumors Using Rt-PCR on Fixed Tissues Useful in Differentiating Histologic Subtypes

2004 ◽  
Vol 171 (4S) ◽  
pp. 436-436
Author(s):  
John A. Petros ◽  
Audry N. Schuetz ◽  
Andrew N. Young ◽  
Q. Yin Goen ◽  
So Dug Lim ◽  
...  
Keyword(s):  
Gene Expression ◽  
Renal Tumors ◽  
Rt Pcr ◽  
Novel Gene ◽  
Histologic Subtypes
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