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Results for “Relative copy number variation”

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PubMed2005

[Comparison of therapeutic effects of low-dose versus high-dose interferon alpha-2b treatment on chronic myelocytic leukemia: a prospective randomized study].

OBJECTIVE: To compare the therapeutic effects of low-dose and high-dose interferon alpha-2b (IFN) treatment on chronic myelocytic leukemia (CML). METHODS: A real-time quantitative reverse transcriptase PCR (RQ-PCR) method was established to detect the fusion gene bcr-abl expression, thereby studying the reduction of leukemic cells. Thirty newly diagnosed CML patients, 21 males and 9 females, aged 14 - 69, were treated with hydroxyurea to keep the white blood cell count less than 20 x 10(9)/L, and then randomized into 2 groups: high-dose IFN group receiving IFN alpha-2b 5MIU 6 times per week for 3 - 6 months and low-dose IFN group receiving IFN alpha-2b 3MIU every other day for 3 - 6 months. Bone marrow was collected every month to Real-time PCR was used to detect the expression of bcr-abl mRNA. Mononuclear cells were isolated and RNA was extracted to detect the expression of fusion gene bcr-abl and a control gene GAPDH. The results were reported as the number of bcr-abl copies/GAPDH copy. RESULTS: The established real-time quantitative PCR method could detect the bcr-abl molecules as low as 50 copies. The intra-assay coefficient of variation (CV) was less than 5% and the inter-assay CV was 5.13%. The median bcr-abl fusion gene expression level of 30 CML patients before IFN therapy was 0.098 (range: 0.010 - 5.799). The bcr-abl expression level decreased by 19.37% and 24.86% in the low-dose and high-dose IFN groups respectively after 3 months' therapy. No significant difference was observed between the two groups (P = 0.398). Relatively more side effects were observed in the high-dose IFN group than in low-dose group. CONCLUSION: RQ-PCR is a reliable method to monitor CML therapy by analyzing fusion gene bcr-abl expression. There is a difference in bcr-abl fusion gene expression levels among the newly diagnosed patients, and low-dose IFN is as effective as high-dose IFN in reducing bcr-abl expression but with less side effects.

Adolescent↗

PubMed2006

Retrotransposon sequence variation in four asexual plant species.

Transposable elements (TEs) can be viewed as genetic parasites that persist in populations due to their capacity for increase in copy number and the inefficacy of selection against them. A corollary of this hypothesis is that TEs are more likely to spread within sexual populations and be eliminated or inactivated within asexual populations. While previous work with animals has shown that asexual taxa may contain less TE diversity than sexual taxa, comparable work with plants has been lacking. Here we report the results of a study of Ty1/copia, Ty3/gypsy, and LINE-like retroelement diversity in four asexual plant species. Retroelement-like sequences, with a high degree of conservation both within and between species, were isolated from all four species. The sequences correspond to several previously annotated retroelement subfamilies. They also exhibit a pattern of nucleotide substitution characterized by an excess of synonymous substitutions, suggestive of a history of purifying selection. These findings were compared with retroelement sequence evolution in sexual plant taxa. One likely explanation for the discovery of conserved TE sequences in the genomes of these asexual taxa is simply that asexuality within these taxa evolved relatively recently, such that the loss and breakdown of TEs is not yet detectable through analysis of sequence diversity. This explanation is examined by conducting stochastic simulation of TE evolution and by using published information to infer rough estimates of the ages of asexual taxa.

Amino Acid Sequence↗

PubMed1999

A variant Klinefelter syndrome patient with an XXY/XX/XY karyotype studied by GTG-banding and fluorescence in situ hybridization.

Klinefelter syndrome is the first human sex chromosomal abnormality to be reported. The majority of Klinefelter syndrome patients have the XXY karyotype. Approximately 15% of Klinefelter patients, however, are mosaics with variable phenotypes. Among the variant Klinefelter genotypes are such karyotypes as XY/XXY and XX/XXY. The variation in phenotypes most likely depends on the number of abnormal cells and their location in body tissues. In this paper we report the case of a 42-year-old patient with Klinefelter syndrome and a rare variant mosaic XXY/XX karyotype initially identified by GTG-banding. This was confirmed by fluorescence in situ hybridization (FISH) using a dual-color X/Y probe. The patient presented with erectile dysfunction and few other physical findings. Thus, this case illustrates a rare variant of Klinefelter syndrome with a relatively mild phenotype. It also illustrates the utility of FISH as an adjunct to conventional cytogenetics in assessing the chromosome copy number in each cell line of a mosaic. In our case, FISH also detected the presence of a small population of cells with the XY karyotype not previously detected in the initial 30-cell GTG-banding analysis. Thus, through a combination of GTG-banding and FISH, the patient was determined to be an XXY/XX/XY mosaic. Given that most individuals with Klinefelter syndrome are infertile, and that these individuals may wish to reproduce with the aid of modern reproductive technology, such as testicular fine needle aspiration and intracytoplasmic sperm injection, it is important that accurate estimation of the frequency of abnormal cells be obtained for accurate risk estimation and genetic counseling, as recent studies in patients with mosaic Klinefelter syndrome revealed that germ cells with sex chromosomal abnormalities were nevertheless capable of completing meiosis.

Adult↗

PubMed2025

MPAC: a computational framework for inferring pathway activities from multi-omic data.

MOTIVATION: Fully capturing cellular state requires examining genomic, epigenomic, transcriptomic, proteomic, and other assays for a biological sample and comprehensive computational modeling to reason with the complex and sometimes conflicting measurements. Modeling these so-called multi-omic data is especially beneficial in disease analysis, where observations across omic data types may reveal unexpected patient groupings and inform clinical outcomes and treatments. RESULTS: We present Multi-omic Pathway Analysis of Cells (MPAC), a computational framework that interprets multi-omic data through prior knowledge from biological pathways. MPAC leverages network relationships encoded in pathways through a factor graph to infer consensus activity levels for proteins and associated pathway entities from multi-omic data, runs permutation testing to eliminate spurious activity predictions, and groups biological samples by pathway activities to allow identifying and prioritizing proteins with potential clinical relevance, e.g. associated with patient prognosis. Using DNA copy number alteration and RNA-seq data from head and neck squamous cell carcinoma patients from The Cancer Genome Atlas as an example, we demonstrate that MPAC predicts a patient subgroup related to immune responses not identified by analysis with either input omic data type alone. Key proteins identified via this subgroup have pathway activities related to clinical outcome as well as immune cell composition. Our MPAC R package enables similar multi-omic analyses on new datasets. AVAILABILITY AND IMPLEMENTATION: The MPAC package is available at Bioconductor https://bioconductor.org/packages/MPAC.

Humans↗

PubMed2004

Assessment of monitor conditions for the display of radiological diagnostic images and ambient lighting.

Diagnostic efficacy is related to viewing conditions. An increasing number of radiology departments are using workstations for reporting and it was the aim of this study to assess monitor performance and ambient lighting in areas allocated to soft-copy reporting and review. The study was performed in 4 Dublin hospitals and 20 monitors were examined. Using a Society of Motion Pictures and Television Engineers' (SMPTE) test pattern, maximum luminance, spatial uniformity of luminance, temporal luminance stability, brightness and contrast resolution (gamma), geometry and ambient lighting was assessed. The results demonstrated that although temporal luminance stability and spatial uniformity appeared to be at acceptable levels, maximum luminance and gamma value variations were noted, with maximum luminance and geometry values often not complying with published guidelines. Cleaning the monitor face had no impact. 90% of viewing areas had acceptable ambient lighting levels. The data presented demonstrate that monitors examined were not operating at optimal levels for all performance parameters and inclusion of regular assessments of monitors should be part of an imaging department's ongoing quality assurance programme.

Computer Terminals↗

PubMed2025

Regulation of replication timing in Saccharomyces cerevisiae.

In order to maintain genomic integrity, DNA replication must be highly coordinated. Disruptions in this process can cause replication stress which is aberrant in many pathologies including cancer. Despite this, little is known about the mechanisms governing the temporal regulation of DNA replication initiation, thought to be related to the limited copy number of firing factors. Here, we present a high (1-kilobase) resolution stochastic model of Saccharomyces cerevisiae whole-genome replication in which origins compete to associate with limited firing factors. After developing an algorithm to fit this model to replication timing data, we validated the model by reproducing experimental inter-origin distances, origin efficiencies, and replication fork directionality. This suggests the model accurately simulates the aspects of DNA replication most important for determining its dynamics. We also use the model to predict measures of DNA replication dynamics which are yet to be determined experimentally and investigate the potential impacts of variations in firing factor concentrations on DNA replication.

Saccharomyces cerevisiae↗

PubMed2002

A small reservoir of disabled ORFs in the yeast genome and its implications for the dynamics of proteome evolution.

We surveyed the sequenced Saccharomyces cerevisiae genome (strain S288C) comprehensively for open reading frames (ORFs) that could encode full-length proteins but contain obvious mid-sequence disablements (frameshifts or premature stop codons). These pseudogenic features are termed disabled ORFs (dORFs). Using homology to annotated yeast ORFs and non-yeast proteins plus a simple region extension procedure, we have found 183 dORFs. Combined with the 38 existing annotations for potential dORFs, we have a total pool of up to 221 dORFs, corresponding to less than approximately 3% of the proteome. Additionally, we found 20 pairs of annotated ORFs for yeast that could be merged into a single ORF (termed a mORF) by read-through of the intervening stop codon, and may comprise a complete ORF in other yeast strains. Focussing on a core pool of 98 dORFs with a verifying protein homology, we find that most dORFs are substantially decayed, with approximately 90% having two or more disablements, and approximately 60% having four or more. dORFs are much more yeast-proteome specific than live yeast genes (having about half the chance that they are related to a non-yeast protein). They show a dramatically increased density at the telomeres of chromosomes, relative to genes. A microarray study shows that some dORFs are expressed even though they carry multiple disablements, and thus may be more resistant to nonsense-mediated decay. Many of the dORFs may be involved in responding to environmental stresses, as the largest functional groups include growth inhibition, flocculation, and the SRP/TIP1 family. Our results have important implications for proteome evolution. The characteristics of the dORF population suggest the sorts of genes that are likely to fall in and out of usage (and vary in copy number) in a strain-specific way and highlight the role of subtelomeric regions in engendering this diversity. Our results also have important implications for the effects of the [PSI+] prion. The dORFs disabled by only a single stop and the mORFs (together totalling 35) provide an estimate for the extent of the sequence population that can be resurrected readily through the demonstrated ability of the [PSI+] prion to cause nonsense-codon read-through. Also, the dORFs and mORFs that we find have properties (e.g. growth inhibition, flocculation, vanadate resistance, stress response) that are potentially related to the ability of [PSI+] to engender substantial phenotypic variation in yeast strains under different environmental conditions. (See genecensus.org/pseudogene for further information.)

Chromosomes, Fungal↗

PubMed2026

Mitochondrial Haplotype Shapes the Trajectory of Ovarian Aging in Genetically Heterogeneous Rats.

Ovarian aging leads to permanent reproductive senescence and systemic hormonal changes that predispose women to age-associated comorbidities. Despite these observations, the intrinsic mechanisms driving age-related ovarian decline are poorly defined. Mitochondrial DNA (mtDNA) mutations and instability are strongly associated with aging; however, it remains unknown if naturally occurring mitochondrial genetic variation influences the trajectory of ovarian aging. To address this, we compared two genetically heterogeneous rat cohorts (OKC-HETB and OKC-HETW) that differ in mitochondrial haplotype on a randomized but equivalently distributed nuclear background. The OKC-HETW haplotype was associated with accelerated loss of primordial follicles and pathological remodeling marked by fibrosis, macrophage infiltration, and multinucleated giant cells. These tissue-level pathologies were paralleled by mitochondrial dysfunction, characterized by decreased respiratory complex activity, ATP production, and mtDNA copy number. Mechanistically, we identified a haplotype-specific defect in mitochondrial genome maintenance. Although TFAM expression was normal, and total TFAM protein was elevated, OKC-HETW ovaries showed reduced mitochondrial TFAM abundance, TFAM-mtDNA binding, and TOMM20, suggesting that impaired TOMM20-mediated import is associated with compromised mitochondrial genomic stability. Longitudinal transcriptomic and proteomic analyses further indicate that mitochondrial haplotype influences the rate of ovarian aging, with OKC-HETW ovaries showing accelerated activation of inflammatory and fibrotic pathways alongside suppressed proteostasis and mitochondrial function. These defects corresponded to impairments in ovulation and a trend toward worsening oocyte quality. Collectively, our findings identify mitochondrial haplotype as a heritable modifier of ovarian aging rate that acts in concert with the nuclear genome, and a putative target for preserving ovarian function and female healthspan.

Animals↗

PubMed1985

Comparison of MHC genes among distantly related members of the genus Mus.

The genomic content of class I and class II MHC DNA sequences in a variety of wild mice has been analyzed. The panel of mice includes members of three subgenera of the genus Mus. By genomic hybridization with the use of a variety of DNA probes, both class I and class II DNA sequences appear to be conserved in all of the species examined. However, the number of class I DNA sequences differs among the species. Furthermore, this variation appears to result from differential increases within subsets of class I genes. These data suggest that the class I multigene family is dynamic and changing over short periods of evolutionary time. In contrast, none of the class II genes appears to vary in copy number. More extensive polymorphism was noted amongst the class II beta genes than the alpha genes. Interestingly, the genomic sequence corresponding to E beta 2 is highly conserved, leading to the prediction that it is a genetically functional sequence.

Animals↗

PubMed2006

Ribosomal DNA in the grasshopper Podisma pedestris: escape from concerted evolution.

Eukaryote nuclear ribosomal DNA (rDNA) typically exhibits strong concerted evolution: a pattern in which several hundred rDNA sequences within any one species show little or no genetic diversity, whereas the sequences of different species diverge. We report a markedly different pattern in the genome of the grasshopper Podisma pedestris. Single individuals contain several highly divergent ribosomal DNA groups. Analysis of the magnitude of divergence indicates that these groups have coexisted in the Podisma lineage for at least 11 million years. There are two putatively functional groups, each estimated to be at least 4 million years old, and several pseudogene groups, many of which are transcribed. Southern hybridization and real-time PCR experiments show that only one of the putatively functional types occurs at high copy number. However, this group is scarcely amplified under standard PCR conditions, which means that phylogenetic inference on the basis of standard PCR would be severely distorted. The analysis suggests that concerted evolution has been remarkably ineffective in P. pedestris. We propose that this outcome may be related to the species' exceptionally large genome and the associated low rate of deletion per base pair, which may allow pseudogenes to persist.

Animals↗

PubMed1984

Variation in V lambda genes in the genus Mus.

The complement of Ig V lambda genes in nine species of feral mice representing the four extant subgenera of the genus Mus was examined and compared with that of BALB/c inbred mice. Although all inbred strains examined have two V lambda genes, there is variation in the number of copies of V lambda genes in the wild mice. All feral representatives of M. musculus domesticus, from which inbred strains are derived, have at least three V lambda genes, indicating that a V lambda gene may have been lost during the inbreeding process. At least three V lambda genes are also found in representatives of three other M. musculus subspecies, including the stock of M. musculus musculus "Czech II" shown to have at least 12 C lambda genes. In comparing the complement of V lambda and C lambda genes in these animals, evidence is found that supports a mechanism of lambda gene reiteration involving duplication of a unit containing a V lambda and two C lambda genes. However, the possibility that C lambda gene amplification occurred independent of V lambda gene evolution cannot be ruled out. M. spicelegus and M. spretus, species that are semifertile with M. musculus, have one to three V lambda genes. Species more distantly related to M. musculus, such as M. cookii and M. platythrix, appear to have more (four to six) V lambda genes. Greater V lambda gene heterogeneity is also found in these animals. We propose that the ancestors of the subgenus Mus had more V lambda genes than are seen in modern species and that the paucity of V lambda genes in M. musculus, M. spicelegus, and M. spretus may be the result of V lambda gene deletion events that occurred since the divergence of the ancestor of these three species and those of the distantly related species.

Animals↗

PubMed2006

Beyond HLA: the significance of genomic variation for allogeneic hematopoietic stem cell transplantation.

The last 2 years have seen much excitement in the field of genetics with the identification of a formerly unappreciated level of "structural variation" within the normal human genome. Genetic structural variants include deletions, duplications, and inversions in addition to the recently discovered, copy number variants. Single nucleotide polymorphisms are the most extensively evaluated variant within the genome to date. Combining our knowledge from these studies with our rapidly accumulating understanding of structural variants, it is apparent that the extent of genetic dissimilarity between any 2 individuals is considerable and much greater than that which was previously recognized. Clearly, this more diverse view of the genome has significant implications for allogeneic hematopoietic stem cell transplantation, not least in the generation of transplant antigens but also in terms of individual susceptibility to transplant-related toxicities. With advances in DNA sequencing technology we now have the capacity to perform genome-wide analysis in a high throughput fashion, permitting a detailed genetic analysis of patient and donor prior to transplantation. Understanding the significance of this additional genetic information and applying it in a clinically meaningful way will be one of the challenges faced by transplant clinicians in the future.

Genetic Predisposition to Disease↗

PubMed2004

Porcine reproductive and respiratory syndrome virus field isolates differ in in vitro interferon phenotypes.

Type I interferons (IFN-alpha and -beta) play an important role in the innate host defense against viral infection by inducing antiviral responses. In addition to direct antiviral activities, type I IFN serves as an important link between the innate and adaptive immune response through multiple mechanisms. Therefore, the outcome of a viral infection can be affected by IFN induction and the IFN sensitivity of a virus. North American porcine reproductive and respiratory syndrome virus (PRRSV) field isolates were studied with regard to IFN-alpha sensitivity and induction in order to understand the role of type I IFN in PRRSV pathogenesis. PRRSV isolates were differentially sensitive to porcine recombinant IFN-alpha (rIFN-alpha) and varied in their ability to induce IFN-alpha in porcine alveolar macrophages (PAM) cultures as measured by a porcine IFN-alpha specific ELISA on cell culture supernatants. Fifty-two plaques were purified from three PRRSV isolates (numbers 3, 7, and 12) and tested for IFN sensitivity and IFN induction. Plaque-derived populations were composed of heterogeneous populations in terms of IFN-inducing capacity and sensitivity to rIFN-alpha. When macrophages infected with isolates 3, 7, or 12 were treated with polycytidylic acid (polyI:C), IFN-alpha production was enhanced. Cells infected with isolate 3 and treated with polyI:C showed the most consistent and strongest enhancement of IFN-alpha production. It was demonstrated that the relatively low concentrations of IFN-alpha produced by isolate 3 contributed to the enhanced IFN-alpha synthesis in response to polyI:C. Isolates 7 and 12 significantly suppressed the enhanced IFN-alpha production by isolate 3 in polyI:C treated cells. To determine if suppression was at the level of IFN-alpha transcription, quantitative RT-PCR was performed for IFN-alpha mRNA and compared to GAPDH and cyclophilin mRNA quantification. However, the relative number of IFN-alpha transcript copies did not correlate with IFN-alpha protein levels, suggesting a post-transcriptional mechanism of suppression. In summary, these results demonstrate that PRRSV field isolates differ both in IFN-alpha sensitivity and induction. Furthermore, a PRRSV field isolate strongly enhance polyI:C-induced IFN-alpha production in PAM cultures and this priming effect was suppressed by other PRRSV isolates.

Animals↗

PubMed2005

Assessing the fidelity of ancient DNA sequences amplified from nuclear genes.

To date, the field of ancient DNA has relied almost exclusively on mitochondrial DNA (mtDNA) sequences. However, a number of recent studies have reported the successful recovery of ancient nuclear DNA (nuDNA) sequences, thereby allowing the characterization of genetic loci directly involved in phenotypic traits of extinct taxa. It is well documented that postmortem damage in ancient mtDNA can lead to the generation of artifactual sequences. However, as yet no one has thoroughly investigated the damage spectrum in ancient nuDNA. By comparing clone sequences from 23 fossil specimens, recovered from environments ranging from permafrost to desert, we demonstrate the presence of miscoding lesion damage in both the mtDNA and nuDNA, resulting in insertion of erroneous bases during amplification. Interestingly, no significant differences in the frequency of miscoding lesion damage are recorded between mtDNA and nuDNA despite great differences in cellular copy numbers. For both mtDNA and nuDNA, we find significant positive correlations between total sequence heterogeneity and the rates of type 1 transitions (adenine --> guanine and thymine --> cytosine) and type 2 transitions (cytosine --> thymine and guanine --> adenine), respectively. Type 2 transitions are by far the most dominant and increase relative to those of type 1 with damage load. The results suggest that the deamination of cytosine (and 5-methyl cytosine) to uracil (and thymine) is the main cause of miscoding lesions in both ancient mtDNA and nuDNA sequences. We argue that the problems presented by postmortem damage, as well as problems with contamination from exogenous sources of conserved nuclear genes, allelic variation, and the reliance on single nucleotide polymorphisms, call for great caution in studies relying on ancient nuDNA sequences.

Animals↗

PubMed2001

[Genetic instability in cancer tissues analyzed by RAPD PCR].

In five kinds of tumors, total 128 specimens were analyzed by RAPD (random amplified polymorphic DNA) PCR with nine 10-base arbitrary primers for detecting instabilities of DNA and chromosome and screening new molecular markers coupled to putative or unknown oncogenes and/or tumor suppressor genes. Bands representing instabilities have been recovered and purified from agarose and cloned into pCAPs vector, and further labeled by DIG as probes for analysis of Southern blot, Northern blot and Sequencing. Results revealed that sample 5 and 3 of the gastric cancers showed the highest genomic changes and the average detectability in five sorts of cancers was up to at least 40% (42.2%-49.4%), and that there were significant differences in the ability of each primer to detect genomic instability, which ranged from 27% to 68%. Despite the highest detectability of genetic instability (68%) in tumor tissues, primer 2 could produce stable profiles of DNA bands in normal tissue genome with good reproducibility. On the contrary, primer 8 was of the lowest one (27%). Band B of single copy found to be allelic losses in gastric and colon cancers according to RFLP analysis was of a novel sequence and registered by Gen-Bank (Accession Number AF151005). Therefore the genetic instabilities often concentrated on some special locuses of chromosome e.g. repetitive sequences etc. and coupled to carcinogenesis. It was impossible or difficult to get great achievements for cancer treatments with the procedure of gene therapy only to one oncogene or one tumor suppressor gene because the extensive DNA variations occurred during the progression of tumor. RAPD assay connected with other techniques was a good tool for the detection of genomic instabilities and direct screening of some new molecular markers related to tumor suppressor genes or oncogenes.

Base Sequence↗

PubMed2006

Doubling genome size without polyploidization: dynamics of retrotransposition-driven genomic expansions in Oryza australiensis, a wild relative of rice.

Retrotransposons are the main components of eukaryotic genomes, representing up to 80% of some large plant genomes. These mobile elements transpose via a "copy and paste" mechanism, thus increasing their copy number while active. Their accumulation is now accepted as the main factor of genome size increase in higher eukaryotes, besides polyploidy. However, the dynamics of this process are poorly understood. In this study, we show that Oryza australiensis, a wild relative of the Asian cultivated rice O. sativa, has undergone recent bursts of three LTR-retrotransposon families. This genome has accumulated more than 90,000 retrotransposon copies during the last three million years, leading to a rapid twofold increase of its size. In addition, phenetic analyses of these retrotransposons clearly confirm that the genomic bursts occurred posterior to the radiation of the species. This provides direct evidence of retrotransposon-mediated variation of genome size within a plant genus.

Base Sequence↗

PubMed2003

Retroelements, transposons and methylation status in the genome of oil palm (Elaeis guineensis) and the relationship to somaclonal variation.

We isolated and characterized different classes of transposable DNA elements in oil palm (Elaeis guineensis) plants grown from seed, and plants regenerated from tissue culture that show mantling, an abnormality leading to flower abortion. Using PCR assays, reverse transcriptase fragments belonging to LINE-like and gypsy-like retroelements and transposase fragments of En/Spm transposons were cloned. Sequence analysis revealed the presence of a major family of LINEs in oil palm, with other diverged copies. Gypsy-like retrotransposons form a single homologous group, whereas En/Spm transposons are present in several diverged families. Southern analysis revealed their presence in low (LINEs) to medium (gypsy and En/Spm) copy numbers in oil palm, and in situ hybridization showed a limited number of distinct loci for each class of transposable element. No differences in the genomic organization of the different classes of transposable DNA elements between ortet palm (parent) and regenerated palm trees with mantled phenotype were detected, but different levels of sequence methylation were observed. During tissue culture, McrBC digestion revealed the genome-wide reduction in DNA methylation, which was restored to near-normal levels in regenerated trees. HPLC analysis showed that methylation levels were slightly lower in the regenerated trees compared to the ortet parent. The genomic organization of the transposable DNA elements in different oil palm species, accessions and individual regenerated trees was investigated revealing only minor differences. The results suggest that the mantled phenotype is not caused by major rearrangements of transposable elements but may relate to changes in the methylation pattern of other genomic components.

Amino Acid Sequence↗

PubMed1997

Two variants of quantitative reverse transcriptase PCR used to show differential expression of alpha-, beta- and gamma-fibrinogen genes in rat liver lobes.

Quantitative reverse transcriptase PCR (RT-PCR) is a sensitive method for the measurement of mRNA copy number. However, the methodology has gained a reputation for poor reproducibility, leading to concern over the validity of much of the data generated using this technique. We have developed two variants of quantitative competitive RT-PCR using a synthesized RNA as an internal standard to measure precisely the relative levels of alpha-, beta- and gamma-fibrinogen mRNAs in the four lobes of the rat liver. In the first of these variants we altered only the amount of total RNA in the RT-PCR reaction, keeping the amount of internal standard RNA and the number of PCR cycles constant. In the second variant only the number of PCR cycles was altered, and the amounts of total RNA and standard RNA were kept constant. Both variants of RT-PCR allowed calculation of the number of mRNA copies, which did not differ significantly between the two techniques. Of the two variants, the second gave better reproducibility, and the intra-assay coefficient of variation for this technique was 14% (n = 20). Using these two variants we have shown that there are different numbers of fibrinogen mRNAs in the four liver lobes for each of the three genes (alpha-fibrinogen F = 14.64, P = 0.0003; beta-fibrinogen F = 3.74, P = 0.04; gamma-fibrinogen F = 3.75, P = 0.04). In conclusion, by using two variants of quantitative competitive RT-PCR we have shown that this technique can be used to give reproducible results, and the low intra-assay coefficient of variation suggests that quantitative RT-PCR should be the technique of choice for accurate measurement of mRNA copy number.

Animals↗