Preparation of Polymerase Chain Reaction Templates from Embryonic and Adult Mouse Tissue Samples

2019 ◽  
Vol 2019 (3) ◽  
pp. pdb.prot094391
Author(s):  
Janet Rossant ◽  
Andras Nagy
Keyword(s):  
Polymerase Chain Reaction ◽  
Adult Mouse ◽  
Mouse Tissue ◽  
Chain Reaction ◽  
Tissue Samples ◽  
Polymerase Chain ◽  
Adult Mouse Tissue

scholarly journals Development of Quantitative Real-Time Polymerase Chain Reaction for Detection of and Discrimination between Erysipelothrix Rhusiopathiae and Other Erysipelothrix Species

2009 ◽  
Vol 21 (5) ◽  
pp. 701-706 ◽  
Author(s):  
Ho To ◽  
Tomohiro Koyama ◽  
Shinya Nagai ◽  
Kotaro Tuchiya ◽  
Tetsuo Nunoya
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Limit Of Detection ◽  
Enrichment Culture ◽  
Bacterial Species ◽  
Erysipelothrix Rhusiopathiae ◽  
Chain Reaction ◽  
Tissue Samples ◽  
Practical Applications ◽  
Polymerase Chain

Quantitative real-time polymerase chain reaction (qPCR) assays were developed and validated in combination with enrichment culture for the detection and discrimination of Erysipelothrix rhusiopathiae and other Erysipelothrix species from tissue samples. The targets for SYBR green qPCR assays were the 16S ribosomal RNA gene for Erysipelothrix species and a gene involved in capsular formation for E. rhusiopathiae. The specificity of the assays was assessed with Erysipelothrix species and other related bacterial species. The limit of detection was found to be 5 colony-forming units per reaction. Amplification of DNA extracted from spleen and joint samples spiked with increasing quantities of Erysipelothrix cells was shown to be equally sensitive to DNA extracted from a pure bacterial culture. The assays were evaluated with 88 tissue samples from 3 experimentally infected pigs and 50 mice and with 36 tissue samples from 3 naturally infected pigs and 11 noninfected pigs. Results were compared with those of direct qPCR and conventional culture. The qPCR after enrichment increased the diagnostic sensitivity over that of culture and qPCR, thereby significantly reducing the total time taken for the detection of E. rhusiopathiae and other Erysipelothrix species. Therefore, this technique could be used for practical applications.

A Microdissection and Molecular Genotyping Assay to Confirm the Identity of Tissue Floaters in Paraffin-Embedded Tissue Blocks

2003 ◽  
Vol 127 (2) ◽  
pp. 213-217 ◽  
Author(s):  
Jennifer L. Hunt ◽  
Patricia Swalsky ◽  
E. Sasatomi ◽  
Laura Niehouse ◽  
Anke Bakker ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Tissue Sample ◽  
Small Samples ◽  
Allelic Loss ◽  
Chain Reaction ◽  
Neoplastic Tissue ◽  
Tissue Samples ◽  
Genotyping Assay ◽  
Tissue Identification ◽  
Polymerase Chain

Abstract Context.—A recurring problem in surgical pathology practice is specimen mix-up and floater contamination. While many cases can be resolved histologically, a significant number remain unclear and may have serious clinical and medicolegal implications. Objectives.—To design a microdissection and genotyping assay to identify contaminating floater tissues in paraffin-embedded tissues that is optimized for small samples, and to use the assay to resolve a series of clinical cases with floater tissues. Materials and Methods.—Twenty-one cases of possible tissue floater contamination in paraffin-embedded tissue blocks were included. Using 4 unstained, 4-μm-thick histologic sections, multiple sites were microdissected under direct visualization either by hand or by laser capture microdissection. Nonneoplastic and neoplastic tissues were sampled. Polymerase chain reaction was performed for a panel of 10 polymorphic microsatellite markers at 1p34, 3p26, 5q21, 9p21, 10q23, and 17p13. Allele size and content were analyzed semiquantitatively by fluorescent capillary electrophoresis, and the genotypes for the tissues in the paraffin-embedded tissue blocks were compared for identity. Results.—Tissue identification was successful in all cases, despite small tissue sample size and fixation effects. Comparative analysis of neoplastic tissue floaters and the presumptive source tumor was performed when possible to control for possible allelic loss or microsatellite instability. Conclusions.—Microdissection and genotyping are effective and reliable means to objectively resolve problems of possible floater contamination. Even minute tissue samples provide sufficient DNA template for polymerase chain reaction microsatellite analysis. Because of the potential clinical implications of floaters, we recommend that all suspected floaters that would change a diagnosis from benign to malignant be subjected to genotyping assay to confirm the identity of the floater tissue.

Two Novel Nonradioactive Polymerase Chain Reaction-Based Assays of Dried Blood Spots, Genomic DNA, or Whole Cells for Fast, Reliable Detection of Z and S Mutations in the α1-Antitrypsin Gene

1992 ◽  
Vol 38 (10) ◽  
pp. 2100-2107 ◽  
Author(s):  
B S Andresen ◽  
I Knudsen ◽  
P K Jensen ◽  
K Rasmussen ◽  
N Gregersen
Keyword(s):  
Polymerase Chain Reaction ◽  
Genomic Dna ◽  
Dried Blood Spots ◽  
Chain Reaction ◽  
Tissue Samples ◽  
Whole Cells ◽  
Blood Spots ◽  
Allele Specific ◽  
Polymerase Chain ◽  
Alpha 1 Antitrypsin

Abstract Two new nonradioactive polymerase chain reaction (PCR)-based assays for the Z and S mutations in the alpha 1-antitrypsin gene are presented. The assays take advantage of PCR-mediated mutagenesis, creating new diagnostic restriction enzyme sites for unambiguous discrimination between test samples from individuals who are normal, heterozygous, or homozygous for the mutations. We show that the two assays can be performed with purified genomic DNA as well as with boiled blood spots. The new assays were validated by parallel testing with a technique in which PCR is combined with allele-specific oligonucleotide (ASO) probes. In all cases tested the results obtained by the different techniques were in accordance. The new assays can be used for prenatal diagnostics and can be performed directly with boiled tissue samples. Because the new assays are easy to perform and reliable, we conclude that they are well suited for routine diagnosis.

scholarly journals Differential Expression of Cholecystokinin A Receptor in Gallbladder Cancer in the Young and Elderly Suggests Two Subsets of the Same Disease?

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Hasan Raza Kazmi ◽  
Abhijit Chandra ◽  
Kavita Baghel ◽  
Anshuman Singh ◽  
Jaya Nigam ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Gallbladder Cancer ◽  
Differential Expression ◽  
Clinicopathological Parameters ◽  
Enzyme Linked Immunosorbent Assay ◽  
Chain Reaction ◽  
Tissue Samples ◽  
Age Related ◽  
Poorly Differentiated ◽  
Polymerase Chain

Background. Cholecystokinin type A receptor (CCKAR) is known to be overexpressed in variety of human malignancies but information regarding its expression in gallbladder cancer (GBC) is limited. Attempts were now made to investigate expression pattern of CCKAR mRNA and protein in controls and GBC patients and correlate it with various clinicopathological parameters following surgical resection.Materials and Methods. Gallbladder tissue samples from 64 subjects (GBC: 39; control: 25) were studied. Expression of CCKAR mRNA was evaluated by reverse transcriptase-polymerase chain reaction and confirmed using real-time polymerase chain reaction. Protein expression was studied by enzyme-linked immunosorbent assay.Results. Significantly higher expression of CCKAR mRNA(P<0.0001)and protein(P<0.0001)was observed in GBC tissues. Overexpression was also observed for stage III and in moderately and poorly differentiated tumors. When the clinicopathological parameters were compared, we found age dependent decrease in CCKAR expression. Relatively higher expression of CCKAR was observed in younger patients (age < 45 years) having more aggressive disease when compared with elderly ones (age ≥ 45 years).Conclusions. Age related differential expression of CCKAR in GBC may suggest two possible variants of the disease in this endemic belt.

A comprehensive search for microbial DNA and RNA in sarcoidosis tissue samples by quantitative polymerase chain reaction

2017 ◽  
Vol 49 (8) ◽  
pp. 625-627
Author(s):  
Fumio Terasaki ◽  
Hitomi Fukumoto ◽  
Ryo Kawata ◽  
Yoshinobu Hirose ◽  
Shu-ichi Fujita ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Quantitative Polymerase Chain Reaction ◽  
Chain Reaction ◽  
Tissue Samples ◽  
Dna And Rna ◽  
Comprehensive Search ◽  
Polymerase Chain ◽  
Microbial Dna

scholarly journals Dengue, chikungunya and zika virus coinfection: results of the national surveillance during the zika epidemic in Colombia

2019 ◽  
Vol 147 ◽  
Author(s):  
Marcela Mercado-Reyes ◽  
Jorge Acosta-Reyes ◽  
Edgar Navarro-Lechuga ◽  
Sherill Corchuelo ◽  
Angélica Rico ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Zika Virus ◽  
Medical Records ◽  
National Surveillance ◽  
Chain Reaction ◽  
Zikv Infection ◽  
Tissue Samples ◽  
Epidemiologic Surveillance ◽  
Polymerase Chain

AbstractOur objective was to determine the frequency of zika (ZIKV), chikungunya (CHIKV) and dengue (DENV) virus coinfection and describe the mortality cases that occurred during the epidemiologic surveillance of the ZIKV epidemic in Colombia. We analysed all cases of suspected ZIKV infection that were reported to the National Institute of Health (October 2015–December 2016). DENV, CHIKV and ZIKV RNA were detected in serum or tissue samples using polymerase chain reaction assay. Medical records of the fatal cases were reviewed. We identified that 23 871 samples were processed. The frequency of viral agents was 439 (1.84%) for DENV, 257 (1.07%) for CHIKV and 10118 (42.38%) for ZIKV. Thirty-four (0.14%) cases of coinfection were identified. The CHIKV–ZIKV coinfection was present in 28 cases (82.3%), DENV–CHIKV in three (8.8%) and DENV–ZIKV in three (8.8%). Seven (20.6%) coinfection cases were fatal (two DENV–CHIKV cases and five CHIKV–ZIKV cases). Two cases were foetal deaths and the others were related to neurological syndrome and sepsis. In conclusion, the frequency of arbovirus coinfection during epidemic of ZIKV was low, and CHIKV–ZIKV coinfection was the most common. Mortality was high among coinfection patients. The role of each virus in the mortality cases of coinfection warrants further studies.

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