A Non-Radioactive Method for Inexpensive Quantitative RT-PCR

1999 ◽  
Vol 380 (6) ◽  
pp. 695-697 ◽  
Author(s):  
M. Maggiolini ◽  
O. Donzé ◽  
D. Picard
Keyword(s):  
Gel Electrophoresis ◽  
Agarose Gel Electrophoresis ◽  
Rt Pcr ◽  
Nylon Membrane ◽  
Pcr Product ◽  
Highly Sensitive ◽  
Pcr Products ◽  
Specialized Equipment ◽  
Novel Method ◽  
Direct Incorporation

Abstract We present a novel method for quantitative RT-PCR that involves direct incorporation of digoxigenin-11-dUTP (DIG-dUTP) during amplification of cDNAs, separation of RT-PCR products by agarose gel electrophoresis, Southern transfer to a nylon membrane, and chemiluminescent detection with an anti-DIG antibody. The whole procedure can be done in about a day and has the following advantages: It is highly sensitive, specificity is confirmed by monitoring the size of the RT-PCR product, it is non-radioactive, quantitative, and does not require expensive specialized equipment.

scholarly journals Rapid Detection and Quantification of Rhynchosporium secalis in Barley Using a Polymerase Chain Reaction

2011 ◽  
Vol 42 (No. 3) ◽  
pp. 111-114
Author(s):  
J. Gubiš ◽  
M. Hudcovicová ◽  
M. Gubišová
Keyword(s):  
Gel Electrophoresis ◽  
Pcr Primers ◽  
Rhynchosporium Secalis ◽  
Agarose Gel Electrophoresis ◽  
Artificial Infection ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Green Approach ◽  
Total Dna ◽  
Polymerase Chain

PCR primers for diagnosis of Rhynchosporium secalis in seed samples of barley were developed. For the quantification of the pathogen in seed samples a real-time PCR with SYBR Green approach was used. Amounts from 1.8 to 419.1 pg of R. secalis DNA per 100 ng of total DNA were detected in 18 samples of barley seeds contaminated by R. secalis in field conditions. The correctness of this quantitative analysis was checked using an artificial infection of seeds with 1, 2, 5 and 20% level of infection by R. secalis. The level of contamination of artificially infected samples decreased with a lowering amount of added seed powder contaminated by the pathogen, the correlation coefficient for this analysis was 0.98. While the primer pair used in these analyses shows cross-reactions with other pathogens (P. teres, Drechslera tritici-repentis, F. culmorum and F. poe), it is recommended to check the products of RT-PCR by agarose-gel electrophoresis, in which these pathogens are easily distinguishable from R. secalis by different lengths of the amplified fragments.  

scholarly journals AtypicalCTSKTranscripts andARNTTranscription Read-Through intoCTSK

2005 ◽  
Vol 6 (5-6) ◽  
pp. 268-276
Author(s):  
Fabienne S. Giraudeau ◽  
Jean-Philippe Walhin ◽  
Paul R. Murdock ◽  
Nigel K. Spurr ◽  
Ian C. Gray
Keyword(s):  
Negative Impact ◽  
Cathepsin K ◽  
Chromosome 1 ◽  
Rt Pcr ◽  
Transcription Start ◽  
Aryl Hydrocarbon ◽  
Pcr Product ◽  
Close Proximity ◽  
Pcr Products ◽  
Human Chromosome 1

The aryl hydrocarbon receptor nuclear translocator (ARNT) and cathepsin K (CTSK) genes lie in a tandem head-to-tail arrangement on human chromosome 1. The two genes are in extremely close proximity; the usualCTSKtranscription start site is less than 1.4 kb downstream of the end of the longest reportedARNTtranscript. By generating an RT-PCR product that overlaps both the 3′ end ofARNTand the 5′ end ofCTSK, we show thatARNTtranscripts may extend through theARNT–CTSKintergenic region and progress into theCTSKgene. Furthermore, by using quantitative RT-PCR from several tissues to detect theARNTexpression signature inCTSKintrons, we show thatARNTtranscripts can read through intoCTSKas far asCTSKintron 3, extending approximately 3.7 kb downstream of the end of the longest previously describedARNTmRNA. Given thatARNTandCTSKare expressed in an overlapping range of tissues,ARNTread-through may have a negative impact onCTSKtranscript levels by interfering withCTSKexpression. We also present evidence for novelCTSKtranscripts following sequence analysis ofCTSK-derived ESTs and RT-PCR products. These transcripts show alternate 5′ splicing and or 5′ extension and are sometimes initiated from a cryptic alternative promoter which is upstream of the knownCTSKpromoter and possibly in the 3′ UTR ofARNT.

scholarly journals X- and Y-chromosome-specific variants of the amelogenin gene allow non-invasive sex diagnosis for the detection of pseudohermaphrodite goats

2017 ◽  
Vol 65 (4) ◽  
pp. 500-504 ◽  
Author(s):  
Renáta Fábián ◽  
András Kovács ◽  
Viktor Stéger ◽  
Krisztián Frank ◽  
István Egerszegi ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Sex Reversal ◽  
Agarose Gel Electrophoresis ◽  
Amelogenin Gene ◽  
Non Invasive ◽  
Faecal Samples ◽  
Pcr Products ◽  
Invasive Method ◽  
Pcr Method ◽  
Male Sex

The Polled Intersex Syndrome (PIS) is responsible for the absence of horns in homozygous and heterozygous goats causing a female-to-male sex reversal in the homozygous polled genotypic female (XX) goats. A simple and efficient non-invasive method was elaborated to detect the genotypic sex from hair and faecal samples using a pair of primers to amplify the X- and Y-linked alleles of the amelogenin gene. The PCR products were easily distinguishable using agarose gel electrophoresis: we detected an X-specific single band in samples originating from healthy phenotypic females and double (X- and Y-) bands in samples from males. The new PCR method is applicable for diagnosing the sex of PIS-affected animals already as newborn kids, in contrast with the phenotypic findings appearing only after puberty, and thus it may replace the cumbersome chromosome investigations.

scholarly journals SEX DETERMINATION OF BALI STARLING (Leucopsar rothschildi) USING MOLECULAR SEXING

2015 ◽  
Vol 2 (1) ◽  
pp. 114
Author(s):  
Putu Indra Pramana Wirastika ◽  
Ignatius Pramana Yuda ◽  
Felicia Zahida
Keyword(s):  
Dna Binding ◽  
Sex Determination ◽  
Gel Electrophoresis ◽  
Morphological Characters ◽  
Molecular Sexing ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
Pcr Products ◽  
Conventional Methods

<p>Bali Starling (Leucopsar rothschildi) are monomorphic at the age of nestling. For the conservation of bird it important is to determine its sex at the earlier stage. Conventional methods have limitations. This study applied PCR-based molecular sexing to answer this issue. This study aimed to obtain the most effective molecular primers to identify the sex of Bali starling. The most common used combination of P2/P8, 2550F/ 2718R and 1237L/1272H primers, which amplify CHD1 gene (Chromo-helicase-DNA-binding) were evaluated. DNA samples were obtained from secondary wing feathers of young Bali Starling. Separation in agarose gel electrophoresis of PCR products showed that the three primers were successfully amplified the samples with different degrees of success, that was 90% (P2/P8), 86.7% (2550F/2718R), and 73.3% (1237L/1272H), respectively. However, only the combination of P2/P4 and 2550F/2718R primers was able to sex Bali Starling based on observation of PCR products on agarose gel. The sizes of the genes were slightly different with those reported on previous studies. Most of the results of molecular sexing were in accordance with the sex based on morphological characters.</p><p><br /><strong>Keywords</strong> : Bali starling, Leucopsar rothschildi, molecular sexing, CHD gene</p>

scholarly journals Genetic variability of broodstocks of restocking programs in Brazil

2015 ◽  
pp. 4677-4687 ◽  
Author(s):  
Nelson Lopera-Barrero ◽  
Ed Lima ◽  
Luiz Filho ◽  
Elenice Goes ◽  
Pedro Castro ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Genetic Variability ◽  
Gel Electrophoresis ◽  
Shannon Index ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
General Management ◽  
Rhamdia Quelen ◽  
Pcr Products ◽  
Salminus Brasiliensis

ABSTRACT Objective. The aim of this study was evaluate the genetic diversity of the following broodstocks: piapara (Leporinus elongatus), dourado (Salminus brasiliensis), jundiá (Rhamdia quelen) and cachara (Pseudoplatystoma fasciatum) already useful for restocking programs in the Paranapanema, Iguaçu and Paraná Brazilian Rivers. Materials and methods. Samples from the caudal fin of 122 fish were analyzed. DNA was extracted by NaCl protocol. PCR products were separated by a horizontal agarose gel electrophoresis. The fragments were visualized by staining with ethidium bromide. Results. The amplification of 25 primers generated different fragments in studied species that allowed characterizing 440 fragments of 100-2900 bp. High percentage of polymorphic fragments (66.67 to 86.29), Shannon index (0.365 to 0.486) and genetic diversity of Nei (0.248 to 0.331) were detected. Conclusions. The level of genetic variability in the broodstocks was adequate for allowing their use in restocking programs in the studied Rivers. However, periodical monitoring studies of genetic variability in these stocks, the mating system, reproductive system and general management must be made to guarantee the preservation of wild populations.RESUMEN Objetivo. El objetivo de este estudio fue evaluar la diversidad genética de los siguientes lotes de reproductores: piapara (Leporinus elongatus), dourado (Salminus brasiliensis), jundiá (Rhamdia quelen) y cachara (Pseudoplatystoma fasciatum) utilizados para programas de repoblación en los ríos brasileños Paranapanema, Iguaçu y Paraná. Materiales y métodos. Muestras de aleta caudal de 122 peces fueron analizadas. El ADN fue extraído por el protocolo de NaCl. Los productos de PCR fueron separados por electroforesis horizontal en gel de agarosa. Los fragmentos fueron visualizados por marcación con bromuro de etidio. Resultados. La amplificación de los 25 iniciadores produjo diferentes fragmentos en las especies estudiadas que permitieron caracterizar 440 fragmentos de 100 a 2900 pb. Fueron detectados un alto porcentaje de fragmentos polimórficos (66.67 a 86.29), de índice de Shannon (0.365 a 0.486) y de diversidad genética de Nei (0.248 a 0.331). Conclusiones. El nivel de variabilidad genética en los lotes de reproductores fue adecuado para su utilización en programas de repoblación en los ríos estudiados. Sin embargo, estudios de monitoreo periódico de la variabilidad genética en esos lotes, del sistema de cruzamiento, del sistema reproductivo y del manejo general deben ser realizados para garantizar la preservación de las populaciones naturales. 

Development of a DNA Microarray for the Simultaneous Detection and Genotyping of Noroviruses

2008 ◽  
Vol 71 (7) ◽  
pp. 1434-1441 ◽  
Author(s):  
FRANCO PAGOTTO ◽  
NATHALIE CORNEAU ◽  
KIRSTEN MATTISON ◽  
SABAH BIDAWID
Keyword(s):  
Molecular Epidemiology ◽  
Molecular Characterization ◽  
Simultaneous Detection ◽  
Oligonucleotide Array ◽  
Rt Pcr ◽  
Major Drawback ◽  
Pcr Product ◽  
Reaction Specificity ◽  
Pcr Products

Current methods for detecting and genotyping noroviruses focus on the use of reverse transcriptase (RT)–mediated PCR. A major drawback of this approach is that short target RT-PCR products do not always encompass sequences that can be compared among research laboratories, resulting in difficulties for molecular epidemiology. We describe the use of a microarray-based system for simultaneous detection and molecular characterization of noroviruses. The protocol generates a 917-bp RT-PCR product that encompasses two major regions currently used for detection and analysis of norovirus genomes. The PCR products are then hybridized to an oligonucleotide array (NoroChip) based on 50-mer features, which allows for both confirmation of reaction specificity and molecular characterization of the amplified genome. Parallel sequence analyses of amplicons revealed that our microarray data were robust in separating genogroups I and II, and further subtyping to the cluster level was possible. This approach, combining detection and characterization, overcomes the need for expensive and time-consuming sequence analysis of amplified genome targets for molecular epidemiology.

scholarly journals First Report of Potato spindle tuber viroid in Cape Gooseberry (Physalis peruviana) from Turkey and Germany

Plant Disease ◽  
2009 ◽  
Vol 93 (3) ◽  
pp. 316-316 ◽  
Author(s):  
J. Th. J. Verhoeven ◽  
M. Botermans ◽  
J. W. Roenhorst ◽  
J. Westerhof ◽  
E. T. M. Meekes
Keyword(s):  
Potato Spindle Tuber Viroid ◽  
Rt Pcr ◽  
Physalis Peruviana ◽  
Source Of Infection ◽  
Pcr Product ◽  
Pcr Products ◽  
Mother Plants ◽  
Tomato Chlorotic Dwarf Viroid ◽  
Plant Pathol ◽  
Cape Gooseberry

Since the recent identification of Potato spindle tuber viroid (PSTVd) in vegetatively propagated ornamental plant species (4), many growers have asked to have their mother plants tested for this viroid. In December of 2007, a grower from Turkey submitted cuttings of cape gooseberry (Physalis peruviana) to be tested for PSTVd. Initial testing by real-time reverse transcription (RT)-PCR according to Boonham et al. (1) indicated the presence of either Mexican papita viroid, PSTVd, or Tomato chlorotic dwarf viroid in four samples. To identify the viroid(s) present, isolated RNA from these samples was used for RT-PCR (2), and products of the expected full genome size for the three viroids were amplified from each sample. One of the PCR products was sequenced (GenBank Accession No. EU862230) and analysis of the 357 nt sequence indicated it was most related to PSTVd sequences belonging to the so-called ‘Oceanian’ strain of the viroid (3), with 99.7% identity to GenBank Accession No. AY962324. Therefore, the viroid was identified as PSTVd. Pathogenicity of this PSTVd genotype was demonstrated when 4 weeks after mechanical inoculation with sap extracts seedlings of tomato cv. Money-maker showed the expected viroid symptoms of chlorosis and stunting, and the presence of the viroid in these plants was confirmed by RT-PCR (2). In March of 2008, by use of RT-PCR (2) and sequencing of the PCR product (GenBank Accession No. EU862231), PSTVd was identified in young seedlings of P. peruviana from a German grower. The German isolate differed at only three nucleotide positions from the Turkish isolate. The identification of PSTVd in young seedlings indicates that seeds had been source of infection, whereas in the case of the PSTVd infected cuttings from Turkey, the infection originated from infected mother plants. To our knowledge, these are the first reports of PSTVd in P. peruviana. Although infected P. peruviana plants did not show symptoms, they might act as sources of inoculum for crops like potato and tomato, which may suffer serious damage. References: (1) N. Boonham et al. J. Virol. Methods 116:139, 2004. (2) A. M. Shamloul et al. Can. J. Plant Pathol. 19:89, 1997. (3) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004. (4) J. Th. J. Verhoeven et al. Plant Pathol. 57:399, 2008.

Immunoglobulin Heavy Chain (IgH) Gene Somatic Hypermutation Status Using Homoduplex-Separated RT-PCR Products.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4813-4813
Author(s):  
Melissa A. Melan ◽  
Kenneth A. Foon ◽  
Stanley M. Marks ◽  
Jeffrey A. Kant
Keyword(s):  
Somatic Hypermutation ◽  
Lymphocytic Leukemia ◽  
Rt Pcr ◽  
Mutation Status ◽  
Percent Identity ◽  
Pcr Product ◽  
Pcr Products ◽  
Reverse Primer ◽  
Almost All ◽  
Blast Program

Abstract Assessment of IgH somatic hypermutation status has been shown to be a valuable indicator for judging the prognosis of patients with chronic lymphocytic leukemia (CLL). Our laboratory has developed a streamlined method to improve the rate of successful evaluation of IgH mutation status. Six individual PCR reactions are first performed using random hexamer-generated cDNA as template. These reactions have identical reaction parameters, use a common JH reverse primer and one of six VH class-specific forward primers within Framework 1. PCR products are separated on acrylamide or MetaPhor® agarose gels following formamide denaturation. In almost all cases, a single homoduplex band is resolved indicating a class-specific clonal product. The homoduplex band is excised from the gel, eluted and directly sequenced. Mutation analysis is performed using the NCBI Ig BLAST program with percent identity determined for the region from the beginning of CDR1 to the end of Framework 3. To date, less than 10% of cases analyzed have not yielded a clearcut clonal PCR product using this approach

scholarly journals Immunolocation of Aquaporin 8 in Human Cataractous Lenticular Epithelial Cells

2017 ◽  
Vol 2 (3) ◽  
pp. 1-5 ◽  
Author(s):  
Rijo Hayashi ◽  
Shimmin Hayashi ◽  
Kazunori Fukuda ◽  
Miki Sakai ◽  
Shigeki Machida
Keyword(s):  
Epithelial Cells ◽  
Gel Electrophoresis ◽  
Immunohistochemical Staining ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Anterior Capsule ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Anterior Capsulotomy ◽  
Density Results

Purpose/Aim: Aquaporin 8 (AQP8) is a diffusion facilitator of hydrogen peroxide (H2O2) through cell membranes. The purpose of this study was to confirm and localize AQP8 in human lenticular epithelial cells (LECs). Materials and Methods: Lenticular anterior capsule samples, including LECs, were collected during cataract surgery of cataract patients after informed consent. The localization of AQP8 was detected by immunohistochemical staining using an antibody to AQP8. Real-time polymerase chain reaction (RT-PCR) was also used to determine the AQP8 mRNA expression levels. The PCR products were analyzed by gel electrophoresis following analyses of band density. Results: Immunohistochemical staining showed AQP8 was distributed throughout the whole area of the anterior capsulotomy. AQP8 labeling was observed surrounding and within the cytoplasm of LECs. RT-PCR and gel electrophoresis also revealed the presence of AQP8 mRNA in the lenticular anterior capsule. The results of immunohistochemical staining were comparable to those of RT-PCR and gel electrophoresis. Conclusions: The results of this study indicate the distribution of AQP8 in human LECs. This is the first investigation confirming the presence of AQP8 in human LECs.

Sign in / Sign up

Export Citation Format

Share Document