scholarly journals Automation of Nucleic Acid Isolation on KingFisher Magnetic Particle Processors

2007 ◽  
Vol 12 (4) ◽  
pp. 195-201 ◽  
Author(s):  
Xingwang Fang ◽  
Roy C. Willis ◽  
Angela Burrell ◽  
Kurt Evans ◽  
Quoc Hoang ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Pathogen Detection ◽  
Magnetic Particle ◽  
Rna Isolation ◽  
Viral Rna ◽  
Cell Fractionation ◽  
Rt Pcr ◽  
Total Rna ◽  
Total Rna Isolation

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)

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

2007 ◽  
Vol 38 (1) ◽  
pp. 87-93
Author(s):  
Christian Morsczeck ◽  
Michael Korenkov ◽  
Manfred Nagelschmidt ◽  
Domonkos Feher ◽  
Jörg Michael Schierholz
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Rna Isolation ◽  
Abdominal Hernia ◽  
Rt Pcr ◽  
Total Rna ◽  
Pcr Assays ◽  
Total Rna Isolation

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

2008 ◽  
Vol 38 (2) ◽  
pp. 129-135
Author(s):  
Christian Morsczeck ◽  
Michael Korenkov ◽  
Manfred Nagelschmidt ◽  
Domos Feher ◽  
Jörg Michael Schierholz
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Rna Isolation ◽  
Abdominal Hernia ◽  
Rt Pcr ◽  
Total Rna ◽  
Pcr Assays ◽  
Total Rna Isolation

scholarly journals Isolation and Detection of Enterovirus RNA from Large-Volume Water Samples by Using the NucliSens miniMAG System and Real-Time Nucleic Acid Sequence-Based Amplification

2005 ◽  
Vol 71 (7) ◽  
pp. 3734-3740 ◽  
Author(s):  
Saskia A. Rutjes ◽  
Ronald Italiaander ◽  
Harold H. J. L. van den Berg ◽  
Willemijn J. Lodder ◽  
Ana Maria de Roda Husman
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Large Volume ◽  
Water Samples ◽  
Extraction Method ◽  
Rna Extraction ◽  
Rna Isolation ◽  
Rt Pcr ◽  
Virus Rna ◽  
Nasba Assay

ABSTRACT Concentration of water samples is a prerequisite for the detection of the low virus levels that are present in water and may present a public health hazard. The aim of this study was to develop a rapid, standardized molecular method for the detection of enteroviruses in large-volume surface water samples, using a concentration method suitable for the detection of infectious viruses as well as virus RNA. Concentration of water was achieved by a conventional filter adsorption-elution method and ultrafiltration, resulting in a 10,000-fold concentration of the sample. Isolation of virus RNA by a silica-based RNA extraction method was compared with the nonmagnetic and magnetic NucliSens RNA isolation methods. By using the silica-based RNA extraction method in two out of five samples, enterovirus RNA was detected, whereas four out of five samples were positive following RNA isolation with magnetic silica beads. Moreover, estimated RNA levels increased at least 100 to 500 times. Furthermore, we compared enterovirus detection by an in-house reverse transcription (RT)-PCR with a novel commercially available real-time nucleic acid sequence-based amplification (NASBA) assay. We found that the rapid real-time NASBA assay was slightly less sensitive than our in-house RT-PCR. The advantages, however, of a commercial real-time NASBA assay, like the presence of an internal control RNA, standardization, and enormous decrease in turnaround time, makes it an attractive alternative to RT-PCR.

Per-unit-living tissue normalization of real-time RT-PCR data in ischemic rat hearts

2012 ◽  
Vol 44 (12) ◽  
pp. 651-656 ◽  
Author(s):  
S. Ellefsen ◽  
M. Bliksøen ◽  
A. Rutkovskiy ◽  
I. B. Johansen ◽  
M.-L. Kaljusto ◽  
...  
Keyword(s):  
Infarct Size ◽  
Real Time ◽  
Reference Genes ◽  
Stable Expression ◽  
Unit Weight ◽  
Living Tissue ◽  
Rt Pcr ◽  
Internal Reference ◽  
Total Rna ◽  
Rat Hearts

In studies of gene expression in acute ischemic heart tissue, internal reference genes need to show stable expression per-unit-living tissue to hinder dead cells from biasing real-time RT-PCR data. Until now, this important issue has not been appropriately investigated. We hypothesized that the expression of seven internal reference genes would show stable per-unit-living tissue expression in Langendorff-perfused rat hearts subjected to ischemia-reperfusion. This was found for cyclophilin A, GAPDH, RPL-32, and PolR2A mRNA, with GAPDH showing the highest degree of stability ( R = 0.11), suggesting unchanged rates of mRNA transcription in live cells and complete degradation of mRNA from dead cells. The infarct size-dependent degradation of GAPDH was further supported by a close correlation between changes in GAPDH mRNA and changes in RNA quality measured as RNA integrity number (R = 0.90, P < 0.05). In contrast, β-actin and 18S rRNA showed stable expression per-unit-weight tissue and a positive correlation with infarct size (R = 0.61 and R = 0.77, P < 0.05 for both analyses). The amount of total RNA extracted per-unit-weight tissue did not differ between groups despite wide variation in infarct size (7.1–50.1%). When β-actin expression was assessed using four different normalization strategies, GAPDH and geNorm provided appropriate per-unit-living expression, while 18S and total RNA resulted in marked underestimations. In studies of ischemic tissues, we recommend using geometric averaging of carefully selected reference genes for normalization of real-time RT-PCR data. A marked shift in the mRNA/rRNA ratio renders rRNA as useless for normalization purposes.

Real-Time Nucleic Acid–Based Detection Methods for Pathogenic Bacteria in Food

2004 ◽  
Vol 67 (4) ◽  
pp. 823-832 ◽  
Author(s):  
JOHN L. McKILLIP ◽  
MARYANNE DRAKE
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Food Industry ◽  
Pathogenic Bacteria ◽  
Pathogen Detection ◽  
Food Microbiology ◽  
Detection Methods ◽  
Target Sequence ◽  
Bacterial Enumeration

Quality assurance in the food industry in recent years has involved the acceptance and implementation of a variety of nucleic acid–based methods for rapid and sensitive detection of food-associated pathogenic bacteria. Techniques such as polymerase chain reaction have greatly expedited the process of pathogen detection and have in some cases replaced traditional methods for bacterial enumeration in food. Conventional PCR, albeit sensitive and specific under optimized conditions, obligates the user to employ agarose gel electrophoresis as the means for endpoint analysis following sample processing. For the last few years, a variety of real-time PCR chemistries and detection instruments have appeared on the market, and many of these lend themselves to applications in food microbiology. These approaches afford a user the ability to amplify DNA or RNA, as well as detect and confirm target sequence identity in a closed-tube format with the use of a variety of fluorophores, labeled probes, or both, without the need to run gels. Such real-time chemistries also offer greater sensitivity than traditional gel visualization and can be semiquantitative and multiplexed depending on the specific experimental objectives. This review emphasizes the current systems available for real-time PCR–based pathogen detection, the basic mechanisms and requirements for each, and the prospects for development over the next few years in the food industry.

scholarly journals Comparison of Six RNA Extraction Methods for the Detection of Classical Swine Fever Virus by Real-Time and Conventional Reverse Transcription–PCR

2005 ◽  
Vol 17 (6) ◽  
pp. 574-578 ◽  
Author(s):  
Ming Y. Deng ◽  
He Wang ◽  
Gordon B. Ward ◽  
Tammy R. Beckham ◽  
Thomas S. McKenna
Keyword(s):  
Real Time ◽  
Classical Swine Fever Virus ◽  
Rna Extraction ◽  
Classical Swine Fever ◽  
Extraction Methods ◽  
Rt Pcr ◽  
Total Rna ◽  
Reverse Transcription Pcr ◽  
Rna Extraction Methods ◽  
Positive Results

Six RNA extraction methods, i.e., RNAqueous kit, Micro-to-midi total RNA purification system, NucleoSpin RNA II, GenElute mammalian total RNA kit, RNeasy mini kit, and TRIzol LS reagent, were evaluated on blood and 7 tissues from pig infected with classical swine fever virus (CSFV). Each of the 6 extraction methods yielded sufficient RNA for positive results in a real-time reverse transcription–PCR (RT-PCR) for CSFV, and all RNA, except the one extracted from blood by TRIzol LS reagent, yielded positive results in both a conventional RT-PCR for CSFV and a conventional RT-PCR for an endogenous gene encoding β-actin. The RNA extracted from blood by TRIzol LS reagent became positive in both conventional RT-PCR assays when it was diluted to 1:2, 1:4, or up to 1:64 in nuclease-free water. It is concluded that all 6 methods are more or less useful for the detection of CSFV by real-time and conventional RT-PCR in swine blood and tissues. However, some of the 6 reagents offer certain advantages not common to all 6 extraction procedures. For example, RNA extracted by the TRIzol LS reagent constantly had the highest yield; that by the RNAqueous kit had the highest A260/A280 ratio for almost all samples; and that by the NucleoSpin RNA II and the GenElute mammalian total RNA kit was most likely to be free of contaminations with genomic DNA.

Automated Solutions for Total RNA Isolation from Diverse Sample Types

2006 ◽  
Vol 11 (5) ◽  
pp. 309-313
Author(s):  
P SELLEY ◽  
J BRUNER ◽  
F KELLY ◽  
F MAURIO ◽  
M WATERS ◽  
...  
Keyword(s):  
Rna Isolation ◽  
Total Rna ◽  
Total Rna Isolation
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