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[PCR-diagnostics in investigation of Legionnaires' disease outbreak] Related Articles
[PCR-diagnostics in investigation of Legionnaires' disease outbreak]
Zh Mikrobiol Epidemiol Immunobiol. 2008 Mar-Apr;(2):44-6
Authors:
Results of studies of biological samples from patients with community-acquired pneumonia and samples from environment during Legionnaires' disease outbreak in town Verkhnyaya Pyshma (Sverdlovsk region) in July-August 2007 performed by PCR analysis are presented.
PMID: 18467990 [PubMed - in process]
Identification of Genes Associated with Fumonisin Biosynthesis in Fusarium ve... Related Articles
Identification of Genes Associated with Fumonisin Biosynthesis in Fusarium verticillioides via Proteomics and Quantitative Real-Time PCR.
J Microbiol Biotechnol. 2008 Apr;18(4):648-57
Authors: Choi YE, Shim WB
In this study, we used functional genomic strategies, proteomics and quantitative real-time (qRT)-PCR, to advance our understanding of genes associated with fumonisin production in the fungus Fusarium verticillioides. Earlier studies have demonstrated that deletion of the FCC1 gene, which encodes a C-type cyclin, leads to a drastic reduction in fumonisin production and conidiation in the mutant strain (FT536). The premise of our research was that comparative analysis of F. verticillioides wild-type and FT536 proteomes will reveal putative proteins, and ultimately corresponding genes, that are important for fumonisin biosynthesis. We isolated proteins that were significantly upregulated in either the wild type or FT536 via two-dimensional polyacrylamide gel electrophoresis, and subsequently obtained sequences via mass spectrometry. Homologs of identified proteins, e.g., carboxypeptidase, laccase, and nitrogen metabolite repression protein, are known to have functions involved in fungal secondary metabolism and development. We also identified gene sequences corresponding to the selected proteins and investigated their transcriptional profiles via quantitative real-time (qRT)-PCR in order to identify genes that show concomitant expression patterns during fumonisin biosynthesis. These genes can be selected as targets for functional analysis to further verify their roles in FB1 biosynthesis.
PMID: 18467856 [PubMed - in process]
Technique for quantitative RT-PCR analysis directly from single muscle fibers. Related Articles
Technique for quantitative RT-PCR analysis directly from single muscle fibers.
J Appl Physiol. 2008 May 8;
Authors: Wacker MJ, Tehel MM, Gallagher PM
The use of single cell quantitative RT-PCR has greatly aided the study of gene expression in fields such as muscle physiology. For this study we hypothesized that single muscle fibers from a biopsy can be placed directly into the reverse transcription buffer and gene expression data can be obtained without having to first extract the RNA. To test this hypothesis, biopsies were taken from the vastus lateralis of 5 male subjects. Single muscle fibers were isolated and underwent RNA isolation (technique 1) or placed directly into reverse transcription buffer (technique 2). After cDNA conversion, individual fiber cDNA was pooled and quantitative PCR was performed using primer/probes for beta2 microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), insulin-like growth factor 1 receptor (IGF1R), and glucose transporter subtype 4 (Glut4). The no RNA extraction method provided similar quantitative PCR data as that of the RNA extraction method. A third technique was also tested in which we used (1/4) of an individual fiber's cDNA for PCR (not pooled) and the average coefficient of variation between fibers was less than 8% (CT value) for all genes studied. The no RNA extraction technique was tested on isolated muscle fibers using a gene known to increase after exercise (pyruvate dehydrogenase kinase 4, PDK4). We observed a 13.9 fold change in expression after resistance exercise which is consistent with what has been previously observed. These results demonstrate a successful method for gene expression analysis directly from single muscle fibers. Key words: skeletal muscle, gene expression, Glut4, IGF, PDK4.
PMID: 18467545 [PubMed - as supplied by publisher]
06-Methylguanine-DNA-Methyltransferase in Recurring Anaplastic Ependymomas: P... Related Articles
06-Methylguanine-DNA-Methyltransferase in Recurring Anaplastic Ependymomas: PCR and Immunohistochemistry.
J Chemother. 2008 Apr;20(2):263-8
Authors: Buccoliero AM, Castiglione F, Rossi Degl'innocenti D, Paglierani M, Maio V, Gheri CF, Garbini F, Moncini D, Taddei A, Sardi I, Sanzo M, Giordano F, Mussa F, Genitori L, Taddei GL
Ependymomas are the third most common brain tumor in children. The post surgical management is controversial. There are no convincing data on an effective role for chemotherapy. O(6)-Methylguanine-DNA-Methyltransferase (MGMT) is a DNA repair protein considered to be a chemosensitivity predictor. Hypermethylation of the MGMT gene promoter is an important cause of MGMT inactivation. We evaluated the MGMT gene promoter methylation and the immunohistochemical MGMT protein expression in 12 recurrent anaplastic ependymomas affecting children. Our purpose was to investigate the molecular rationale of the administration of alkylating agents to children affected by recurrent anaplastic ependymomas. All ependymomas lacked MGMT promoter hypermethylation and 9 (75%) showed high MGMT protein expression (>50% tumoral cells). Differences between different recurrences in the same patient were not observed. These results may indicate MGMT as a factor of chemoresistance to alkylating drugs in anaplastic ependymomas and support the uncertainties regarding the actual benefit of chemotherapy for patients with anaplastic ependymomas.
PMID: 18467255 [PubMed - in process]
A kinetic-based sigmoidal model for the polymerase chain reaction and its app... Related Articles
A kinetic-based sigmoidal model for the polymerase chain reaction and its application to high-capacity absolute quantitative real-time PCR.
BMC Biotechnol. 2008 May 8;8(1):47
Authors: Rutledge RG, Stewart D
ABSTRACT: BACKGROUND: Based upon defining a common reference point, current real-time quantitative PCR technologies compare relative differences in amplification profile position. As such, absolute quantification requires construction of target-specific standard curves that are highly resource intensive and prone to introducing quantitative errors. Sigmoidal modeling using nonlinear regression has previously demonstrated that absolute quantification can be accomplished without standard curves; however, quantitative errors caused by distortions within the plateau phase have impeded effective implementation of this alternative approach. RESULTS: Recognition that amplification rate is linearly correlated to amplicon quantity led to the derivation of two sigmoid functions that allow target quantification via linear regression analysis. In addition to circumventing quantitative errors produced by plateau distortions, this approach allows the amplification efficiency within individual amplification reactions to be determined. Absolute quantification is accomplished by first converting individual fluorescence readings into target quantity expressed in fluorescence units, followed by conversion into the number of target molecules via optical calibration. Founded upon expressing reaction fluorescence in relation to amplicon DNA mass, a seminal element of this study was to implement optical calibration using lambda gDNA as a universal quantitative standard. Not only does this eliminate the need to prepare target-specific quantitative standards, it relegates establishment of quantitative scale to a single, highly defined entity. The quantitative competency of this approach was assessed by exploiting "limiting dilution assay" for absolute quantification, which provided an independent gold standard from which to verify quantitative accuracy. This yielded substantive corroborating evidence that absolute accuracies of +/-25% can be routinely achieved. Comparison with the LinReg and Miner automated qPCR data processing packages further demonstrated the superior performance of this kinetic-based methodology. CONCLUSIONS: Called "linear regression of efficiency" or LRE, this novel kinetic approach confers the ability to conduct high-capacity absolute quantification with unprecedented quality control capabilities. The computational simplicity and recursive nature of LRE quantification also makes it amenable to software implementation, as demonstrated by a prototypic Java program that automates data analysis. This in turn introduces the prospect of conducting absolute quantification with little additional effort beyond that required for the preparation of the amplification reactions.
PMID: 18466619 [PubMed - as supplied by publisher]
Screening for PTLD in lung and heart-lung transplant recipients by measuring ... Related Articles
Screening for PTLD in lung and heart-lung transplant recipients by measuring EBV DNA load in bronchoalveolar lavage fluid using real time PCR.
Pediatr Transplant. 2008 Jun;12(4):464-8
Authors: Michelson P, Watkins B, Webber SA, Wadowsky R, Michaels MG
Pediatric L-HLTx recipients are at risk for developing PTLD with the lung being a primary site of disease. We hypothesized that BALF is a better sample than peripheral blood for measuring EBV DNA load in this high-risk population. Archived BALF specimens from pediatric L-HLTx recipients with and without PTLD were assayed for EBV DNA load using a quantitative real time TaqMan PCR assay. These values were compared with values determined in peripheral blood by a competitive PCR assay. Fifty-five BALF specimens from 16 L-HLTx patients were evaluated. Three patients with PTLD had mean BALF EBV DNA load values almost 50-fold higher than subjects without PTLD (4.6 x 10(5) copies/mL vs. 1.0 x 10(4) copies/mL). Patients who were EBV seronegative pretransplantation (i.e., high risk for PTLD) had elevated EBV DNA load values vs. patients who were EBV seropositive pretransplantation, regardless of the diagnosis of PTLD (mean values of 3.2 x 10(5) copies/mL vs. 1.1 x 10(4) copies/mL). Lastly, BALF analysis identified all subjects with PTLD, whereas peripheral blood analysis identified only one of these cases. Therefore, it can be concluded that monitoring EBV DNA load in BALF following L-HLTx facilitates detection of PTLD in high-risk patients and may be superior to peripheral blood assays.
PMID: 18466434 [PubMed - in process]
A Simple Way to Treat PCR Products Prior to Sequencing Using ExoSAP-IT(R). Related Articles
A Simple Way to Treat PCR Products Prior to Sequencing Using ExoSAP-IT(R).
Biotechniques. 2008 May;44(6):834
Authors: Bell J
PMID: 18466158 [PubMed - in process]
A quantitative real-time PCR method for absolute telomere length. Related Articles
A quantitative real-time PCR method for absolute telomere length.
Biotechniques. 2008 May;44(6):807-9
Authors: O'Callaghan N, Dhillon V, Thomas P, Fenech M
Telomere shortening is an important risk factor for cancer and accelerated aging. Here we describe the development of a simple and reproducible method to measure absolute telomere length. Based on Cawthon's quantitative real-time PCR (qRT-PCR) assay, our method uses an oligomer standard that can be used to generate absolute telomere length values rather than relative quantification. We demonstrate a strong correlation between this improved method and the "gold standard" of telomere length measurement[#x02014]terminal restriction fragment analysis (TRF) by Southern hybridization. The capability to generate absolute telomere length values should allow a more direct comparison of results between experiments within and between laboratories.
PMID: 18466151 [PubMed - in process]
Aqueous suspension of carbon nanotubes enhances the specificity of long PCR. Related Articles
Aqueous suspension of carbon nanotubes enhances the specificity of long PCR.
Biotechniques. 2008 Apr;44(4):537-45
Authors: Zhang Z, Shen C, Wang M, Han H, Cao X
DNA manipulation technology is facing more challenges in the postgenomics era. More and more nanomaterials have been investigated for their potential implications in developing better gene technology. In this study, we reported the beneficial effect of single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs) in enhancing the specificity and total efficiency of long (14 kb) PCR. Hydroxylic and carboxylic carbon nanotubes (CNTs) had similar enhancing effects. Nanotubes could become another component for improvements in the amplification of long DNA.
PMID: 18466134 [PubMed - in process]
Performance evaluation of thermal cyclers for PCR in a rapid cycling condition. Related Articles
Performance evaluation of thermal cyclers for PCR in a rapid cycling condition.
Biotechniques. 2008 Apr;44(4):495-505
Authors: Ho Kim Y, Yang I, Bae YS, Park SR
The performance of thermal cyclers for polymerase chain reactions (PCR) is of great concern in terms of the reliability of PCR-based assays, particularly when rapid cycling conditions are applied to small volume reactions. In this work, the precision of the temperature controls during rapid thermal cycling was measured in 19 commercial thermal cyclers of 8 different models. The temperatures of test solutions in specific locations in each thermal block were simultaneously monitored at 1 s intervals during thermal cycling. A temperature-sensitive multiplex PCR was run in parallel to assess undesirable PCR results caused by poor temperature control. Under the given conditions (20 s of annealing time and 20 microL reaction volume), a majority of the tested instruments showed prominent curving, undershooting, and/or overshooting in their temperature profiles, which substantially influenced the results of the temperature-sensitive multiplex PCR. Variations between wells were also observed in most instruments. It is strongly hoped that these problems will be addressed by manufacturers and that they will make substantial improvements in the precision and efficiency of thermal cyclers. In the meantime, users of thermal cyclers might be able to avoid unexpected poor outcomes of sensitive PCR-based assays by designing their PCR protocols with these findings in mind.
PMID: 18466130 [PubMed - in process]
Plastic versus glass capillaries for rapid-cycle PCR. Related Articles
Plastic versus glass capillaries for rapid-cycle PCR.
Biotechniques. 2008 Apr;44(4):487-92
Authors: Elenitoba-Johnson O, David D, Crews N, Wittwer C
Rapid-cycle PCR uses fast temperature transitions and minimal denaturation and annealing times of "0" s to complete 30 cycles in 10 to 30 min. The most popular platform amplifies samples in glass capillaries arranged around a carousel with circulating air for temperature control. Recently, plastic capillary replacements for glass capillaries became available. We compared the performance of plastic and glass capillaries for rapid-cycle PCR. Heat transfer into plastic capillaries was slowed by thicker walls, lower thermal conductivity, and a lower surface area[#x02014]to-volume ratio than glass capillaries. Whereas the denaturation and annealing target temperatures were reached by samples in glass capillaries, samples in plastic capillaries fell short of these target temperatures by 6 degrees [#x02013]7 degrees C. Rapid-cycle PCR was performed on two human genomic targets (APOE and ACVRL1) and one plasmid (pBR322) to amplify fragments of 225[#x02013]300 bp in length with melting temperatures of 90.3 degrees [#x02013]93.1 degrees C. Real-time amplification data, end-point melting curves, and end-point gel analysis revealed strong, specific amplification of samples in glass and complete amplification failure in plastic. Only the APOE target was successfully amplified by extending the denaturation and annealing times to 5 or 10 s. A 20 s holding period was necessary to reach target temperatures in plastic capillaries.
PMID: 18466129 [PubMed - in process]
Sensitive on-chip quantitative real-time PCR performed on an adaptable and ro... Related Articles
Sensitive on-chip quantitative real-time PCR performed on an adaptable and robust platform.
Biomed Microdevices. 2008 May 9;
Authors: Lund-Olesen T, Dufva M, Dahl JA, Collas P, Hansen MF
A robust, flexible and efficient system for performing high sensitivity quantitative on-chip real-time PCR for research purposes is presented. The chips used consist of microchannels etched in silicon. The surface in the channels is a thermally grown silicon dioxide and the channel is sealed by a glass lid. The chips contain four PCR chambers but this number can be increased for further multiplexing. Contrary to PCR chips with oil covered open chambers, these channel-like chambers are easily integrated in lab-on-a-chip devices. The temperature is controlled by a Peltier element and the fluorochrome detector system is a commercially available fluorescence stereo microscope equipped with a CCD camera. The setup shows an excellent signal-to-noise ratio of about 400 compared to that of about 150 obtained in a commercial real time PCR machine. A detection limit of a few copies of target molecules is found, which is 100 to 100,000-fold better than other on-chip real-time PCR systems presented in the literature. This demonstrates that the PCR system can be used for critical applications. We also demonstrate that high quality melting curves can be obtained. Such curves are important in lab-on-a-chip systems for identification of amplified product. The usability of the system is validated by performing quantitative on-chip measurements of the amount of specific gene sequences co-immunoprecipitated with various posttranslationally modified histone proteins. Similar results are obtained from on-chip experiments and experiments carried out in a commercial system on larger sample volumes.
PMID: 18465251 [PubMed - as supplied by publisher]