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Results for “Quantitative PCR”

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At least 19 recordsLinked to original sources
PubMed1992

Accumulation of deletions in human mitochondrial DNA during normal aging: analysis by quantitative PCR.

We have developed a quantitative PCR technique to measure the amount of a specific mitochondrial DNA deletion (delta mtDNA), the so-called 'common deletion', in human tissues. Using this method, we estimate that there is a 10,000-fold increase in this delta mtDNA species in muscle during the course of the normal human lifespan. The maximum amount of common deletion observed in aged muscle was approx. 0.1%. Tissues that turn-over slowly, such as skeletal muscle and heart, contained more delta mtDNA than more rapidly dividing tissues, such as liver, in agreement with studies performed by others.

Aging↗
PubMed1991

Generation of competitor DNA fragments for quantitative PCR.

A convenient and generally applicable method for the generation of competitor DNA fragments for quantitative PCR is described. Using mouse-specific primers, fragments are amplified from DNA of an evolutionarily distantly related species under low-stringency annealing conditions. Because these artificially created fragments contain the mouse primer specific ends, they can be used for the quantification of the mouse DNA amplified by these primers. Competitor DNA fragments that differ in size from the corresponding mouse DNA are selected to distinguish both fragments visually by gel electrophoresis. Competitor DNA fragments were generated for mouse beta-actin, interleukin-1, and tumor necrosis factor (TNF). Co-amplification of beta-actin cDNA for adjustment of equal amounts of input cDNA and subsequently TNF cDNA from lipopolysaccharide (LPS)-activated and nonactivated spleen cells with serial dilutions of the respective competitor DNA fragments allowed a semiquantitative comparison of the ratio of TNF mRNA present in both cDNA samples. Under certain conditions, the competitor DNA fragments can be used to determine the approximate molar concentration of a mRNA.

Animals↗
PubMed1990

Measurement by quantitative PCR of changes in HPRT, PGK-1, PGK-2, APRT, MTase, and Zfy gene transcripts during mouse spermatogenesis.

A reverse transcriptase-polymerase chain reaction assay (RT-PCR) was used quantitatively to measure accumulated levels of RNA transcripts in total mouse RNAs derived from male germ cells at various spermatogenic stages. RNA levels for two X-linked enzymes, phosphoglycerate kinase (PGK-1) and hypoxanthine phosphoribosyl transferase (HPRT), both decrease during spermatogenesis, although the transcript levels decrease much more rapidly for PGK-1. RNA for the Y-linked ZFY (zinc finger protein) is elevated in all spermatogenic cell fractions tested, being particularly high in leptotene/zygotene spermatocytes and round spermatids. RNA for adenine phosphoribosyltransferase (APRT) increases 5-fold to a peak during late pachynema. RNA for PGK-2, undetectable in spermatogonial cells, increases at least 50-fold by the round spermatid stage. DNA (cytosine-5-)-methyltransferase (MTase) transcript levels are over an order of magnitude higher throughout spermatogenesis than in non-dividing liver cells.

Adenine Phosphoribosyltransferase↗
PubMed1992

Analysis of quantitative PCR for the diagnosis of deletion and duplication carriers in the dystrophin gene.

A direct, non-radioactive method of quantitative PCR amplification has been investigated for the diagnosis of deletion and duplication carriers in the dystrophin gene. The simultaneous amplification of two loci, or several loci using multiplex PCR, allows for the direct comparison of relative amounts of products from normal homozygous loci and potentially heterozygous deleted/duplicated loci. Sufficient cycles of PCR are performed to enable visual analysis or densitometric quantification of products on ethidium bromide stained gels. The method has been verified in blind trials performed on known genotypes and by showing that under the conditions used the assay remains within the exponential phase of amplification.

Chromosome Deletion↗
PubMed2026

Development and epidemiological investigation of a TaqMan-based multiplex real-time quantitative PCR assay for simultaneous detection of five bovine viruses (BVDV, AKAV, BNoV, BEV, and BCoV).

INRODUCTION: Infectious diseases caused by bovine viral diarrhea virus (BVDV), Akabane virus (AKAV), bovine norovirus (BNoV), bovine enterovirus (BEV), and bovine coronavirus (BCoV) significantly threaten the cattle industry, resulting in substantial economic losses. These pathogens often present similar clinical signs, such as diarrhea, vomiting, and reproductive disorders in pregnant cattle, and frequent covert or mixed infections further complicate accurate diagnosis. Therefore, rapid, sensitive, and field‑deployable diagnostic methods are essential for effective disease surveillance and control in the cattle industry. METHODS: In this study, we report for the first time the establishment of a TaqMan‑based real‑time quantitative PCR (qPCR) assay that enables simultaneous detection of these five bovine viruses. Multiple sequence alignment of conserved genomic regions was performed, and virus‑specific primers and probes were designed and optimized using Beacon Designer 7 software. Subsequently, a TaqMan‑based multiplex real‑time qPCR assay was established for simultaneous detection of BVDV, AKAV, BNoV, BEV, and BCoV. The established detection method was applied to 200 clinical samples collected from 10 farms in multiple regions of Jilin Province. RESULTS: The results showed that the detection rates for BVDV, AKAV, BNoV, BEV, and BCoV were 33.50%, 0.50%, 4.50%, 7.50%, and 12.00%, respectively. Mixed infections were detected in 9 samples co‑infected with two of the five pathogens, with an overall mixed infection rate of 4.50%. Compared with conventional PCR, coincidence rates were 100% for BVDV, AKAV, BNoV, BEV, and BCoV. DISCUSSION: These findings indicate that the TaqMan multiplex real‑time qPCR assay developed here demonstrates favorable specificity, sensitivity, and reproducibility. This assay enables efficient detection and surveillance of bovine viruses, offering a reliable technical tool for the diagnosis and control of corresponding viral diseases in cattle.

Akabane virus (AKAV)↗
PubMed1991

A general method for quantitative PCR analysis of mRNA levels for members of gene families: application to GABAA receptor subunits.

We have developed a sensitive, PCR-based method for quantitating changes in mRNA levels of members of gene families. In this approach, total mRNA is converted to cDNA and then PCR is carried out on family members simultaneously, using primers derived from regions conserved among family members. This is followed by gel electrophoresis and blotting of the product to filters. The level of expression of individual family members is determined by separate hybridizations using probes unique for each member and derived from sequences between the PCR primers. In this manner the same aliquot of mRNA, the same reverse transcriptase reaction, PCR, gel electrophoresis, and denaturation and blotting are used for analysis of each family member. Thus, experimental variation is minimized, and changes in mRNA levels of family members relative to one another can be monitored with precision. In addition, if a family member is known not to change as a result of the treatment employed, this mRNA can be used to normalize the data from other members and thereby allow individual variations to be quantitated. We have applied this approach to members of the GABAA receptor subunit gene family and studied effects of chronic ethanol treatment on mRNAs corresponding to several GABAA receptor subunits.

Animals↗
PubMed2025

Identification and Analysis of Small Nucleolar RNAs by Real-Time Quantitative PCR.

One of the greatest scientific achievements of the twenty-first century is the completion of The Human Genome Project (HGP). Thereafter, we came to know that the human genome codes nearly 2% for making proteins and thus named as coding genes, suggesting the rest of the genome as noncoding or junk. However, research in the past two decades has shown and established that noncoding RNAs are major contributors of regulating and modulating the various function of cells as well as tissues. Noncoding RNAs can be classified as basis of their sizes in two categories, long noncoding RNAs (>200&#xa0;nt) and small noncoding RNAs (<200&#xa0;nt). Small nucleolar RNAs (snoRNAs) are part of the small noncoding RNA family and primarily reside inside the nucleus of eukaryotes. Sno RNAs can be divided into two major categories based on their distinguished structure and function; these are C/D box and HACA box snoRNAs. They participate in the posttranscriptional modifications on ribosomal RNAs (r-RNAs), transfer RNAs (t-RNAs), messenger RNAs (m-RNAs), and small nuclear RNAs (snRNAs). Sno RNAs act as guide RNAs to modify other noncoding RNAs by pseudouridylation or 2'O ribomethylation. We discussed in this protocol about one of the widely used techniques for detection and analysis of snoRNAs, i.e., real-time quantitative PCR (RT-qPCR).

RNA, Small Nucleolar↗
PubMed2026

Molecular Landscape and Advanced Diagnostic Technologies for BRAF Mutations in Cancer: From Quantitative PCR and ddPCR to CRISPR-Based Platforms.

BRAF mutations are key oncogenic alterations across multiple malignancies, including melanoma, thyroid carcinoma, colorectal cancer, non-small cell lung cancer, glioma, and hairy cell leukemia. The most prevalent variant, BRAF-V600E, induces constitutive activation of the MAPK signaling pathway, promoting tumor progression and influencing therapeutic responsiveness. Accurate detection of BRAF alterations is therefore essential for molecular classification, prognostic assessment, treatment selection, and resistance surveillance. This review summarizes the molecular heterogeneity of BRAF mutations and critically evaluates current diagnostic methodologies. Conventional approaches such as allele-specific PCR and Sanger sequencing are compared with advanced quantitative platforms, including high-resolution melting analysis, droplet digital PCR, and next-generation sequencing, with emphasis on analytical sensitivity, mutation coverage, and clinical applicability. Emerging technologies such as CRISPR-based assays, rolling circle amplification systems, and nanoparticle-based biosensors and point-of-care diagnostic platforms are also discussed for their potential to enhance ultra-sensitive detection, particularly in liquid biopsy settings. These emerging tools are highlighted for their potential to enable ultra-sensitive, rapid, and decentralized mutation detection, particularly in liquid biopsy settings. Key challenges, including intratumoral heterogeneity, low allele-frequency variants, FFPE-associated artifacts, and clonal evolution under therapeutic pressure, are examined within a translational framework. In addition, we examine critical barriers to clinical implementation, including standardization, cost, and global accessibility of molecular diagnostics, and outline potential solutions through scalable technologies and decentralized testing strategies. We propose that optimal BRAF testing requires a mutation subclass-informed and clinically integrated strategy combining comprehensive baseline profiling with longitudinal molecular monitoring. Future diagnostic paradigms will likely integrate multi-omics data and artificial intelligence (AI)-assisted interpretation to refine precision oncology implementation. Looking forward, we propose that optimal BRAF testing will require integration of multi-omics profiling with AI-assisted interpretation, enabling automated variant classification, real-time clinical decision support, and improved prediction of therapeutic response and resistance.

Humans↗
PubMed1991

Molecular diagnosis of sex chromosome aneuploidy using quantitative PCR.

Numeric sex chromosome imbalances, or aneuploidies, are present in several pathological conditions including tumors, abnormal gestations, and clinical syndromes. Here we report a method to identify karyotypic imbalances of the X and Y chromosomes using the polymerase chain reaction (PCR). The polymerase chain reaction was used to quantitatively coamplify the sex chromosome linked genes ZFX and ZFY. Quantitation was facilitated by 1) use of a single primer set which recognizes both templates, 2) incorporation of radiolabelled nucleotides during amplification, and 3) use of amplification conditions which minimize heteroduplex formation. High accuracy of the method was confirmed by concordance with values expected from titrated male and female DNAs and cells from patients with sex chromosome aneuploidy. This approach provides a rapid and reproducible method of evaluating relative abundance of allelic genes, and might be applied to detection of autosomal aneuploidy.

Aneuploidy↗
PubMed1992

A simplified method for determination of specific DNA or RNA copy number using quantitative PCR and an automatic DNA sequencer.

Quantification of specific RNA or DNA molecules that are present in minute amounts in biological samples has previously been performed using PCR in the presence of an internal standard. We have adapted this concept by introducing several modifications that facilitate the quantification of the products and obviate the need for radioisotopes. After amplification, individual products are separated on sequencing gels and directly quantified using a fluorescent automated DNA sequencer. We describe two applications of this approach: the quantitation of minute amounts of bcr-abl hybrid mRNA from malignant cells and the determination of gene copy number in cells stably transfected with a plasmid bearing a chloramphenicol acetyltransferase gene.

Automation↗
PubMed1992

Measurement of mRNA by quantitative PCR with a nonradioactive label.

This report describes the development of a method to measure mRNA in small samples of human tissue by the polymerase chain reaction with a nonradioactive label. In this method RNA is reverse-transcribed in the presence of a control RNA, and subsequently amplified by the polymerase chain reaction during which a nonradioactive label (digoxigenin-11-dUTP) is incorporated. Gel blotting and immunological detection of digoxigenin followed by a chemiluminescent reaction provide an intense signal on film. This allows the detection and quantitation of 3-hydroxy-3-methylglutaryl (HMG) CoA reductase mRNA in 12 ng of RNA. We demonstrate that this is a sensitive and reproducible method, and that quantitation is linear with respect to the amount of mRNA present. The application of this method to the measurement of low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA levels in circulating peripheral blood mononuclear cells and human liver biopsy samples is discussed. The use of chemiluminescent reagents instead of radioactive labels allows this procedure to be performed safely in laboratories not equipped for radioactivity.

Base Sequence↗
PubMed2025

Absolute Quantification of Cellular and Cell-Free Mitochondrial DNA Copy Number from Human Blood and Urinary Samples Using Real Time Quantitative PCR.

Mitochondrial DNA copy number (mtDNA-CN) in human body fluids is widely used as a biomarker of mitochondrial dysfunction in common metabolic diseases. Here we describe protocols to measure cellular and/or cell free (cf)-mtDNA-CN in human peripheral blood and urine. Cellular mtDNA is located inside the mitochondria where it encodes key subunits of the respiratory complexes in mitochondria and is usually normalized with reference to the nuclear genome as the mitochondrial genome to nuclear genome ratio (Mt/N) in either whole blood, peripheral blood mononuclear cells (PBMCs), or whole urine. Cf -mtDNA is usually found outside of the mitochondria, often released following mitochondrial damage, can trigger inflammatory pathways, and is usually measured as mtDNA-CN per volume of the starting material. Here we describe how to (1) separate whole blood into PBMCs, plasma, and serum fractions and whole urine into urinary supernatant and pellet, (2) prepare DNA from each of these fractions, (3) prepare reference&#xa0;standards&#xa0;for absolute quantification, (4) carry out qPCR for either relative or absolute quantification from test samples, (5) analyze qPCR data, and (6) calculate the sample size to adequately power studies. The protocol presented here is suitable for high throughput use and can be modified to quantify mtDNA from other body fluids, human cells, and tissues.

Humans↗
PubMed1992

Characterization of HPV-16 E6/E7 transcription in CaSki cells by quantitative PCR.

Human papillomavirus (HPV) is associated with specific benign and malignant lesions of the epithelial and mucosal surfaces. Of the sexually transmitted types, HPV type 16 (HPV-16) is the most commonly associated with carcinoma of the uterine cervix. Expression of the E6/E7 open reading frame of the viral genome is considered critical in the development of neoplasia. Using the CaSki cervical carcinoma cell line as a model system, we have adapted the polymerase chain reaction to quantify the transcripts expressed from this region. It was found that 97.1% of the total spliced transcript is E6*I, which putatively encodes the E7 oncoprotein, while E6*II comprises 2.9% of spliced product. The ratio of E6*I to E6*II expression may be an important parameter in evaluating the disease risk associated with HPV-16 infection.

Base Sequence↗
PubMed1992

Quantitative PCR with internal controls.

We examine the use of internal controls for estimating the expected initial copy number of the target in a polymerase chain reaction (PCR). We base our investigation on an extended branching-process model. In terms of that model, we delineate the necessary assumptions for this methodology to yield approximately unbiased answers, and we provide means for testing some of those assumptions. We show how to design a series of PCRs to attain optimal precision of the estimate. We provide an algorithm for conducting the statistical analysis of the data, including a formula for a confidence interval for the unknown expected initial copy number.

Algorithms↗
PubMed2026

Testing for Bacteria and Fungi in Cells and Ancillary Reagents-Probe-Based Quantitative PCR Assays.

This document specifies the technical elements for bacteria and fungi detection by PCR assay which enables rapid, broad-spectrum detection of bacteria and fungi with high sensitivity. Qualitative judgement is based on LOD. Key detection points include efficient nucleic acid extraction, broad-spectrum primer-probe design, high amplification efficiency and minimised background interference.

Editorial↗