Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “total RNA”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 793 records · Page 44Linked to original sources
PubMed1994

m1-m5 muscarinic receptor distribution in rat CNS by RT-PCR and HPLC.

Five muscarinic receptor genes (m1-m5) that encode distinct muscarinic receptor subtypes have been cloned. Because of their structural homology and pharmacological similarity, ligand binding probes currently available do not clearly distinguish among the subtypes. To obtain a clear distribution within the CNS of molecularly defined muscarinic receptor subtypes, seven brain regions were examined for the expression of the respective mRNAs. The most sensitive method for detecting mRNA is through amplification of the respective cDNAs. Brain regions were obtained from male Wistar rats, and total RNA was isolated. The isolates were extensively treated with RNase-free DNase to remove any residual genomic DNA. Total RNA (1 microgram) was reverse-transcribed using random primers and reverse transcriptase. The resulting cDNA was amplified using a thermal cycler, and the polymerase chain reaction (PCR)-amplified products were analyzed by gel electrophoresis containing ethidium bromide and visualized with fluorescent illumination. PCR-amplified samples were also injected directly onto an HPLC anion exchange column and quantified by UV detection. Each of the five muscarinic subtypes was found in every brain region examined. The m1 subtype was most abundant in cortex and gradually declined in content caudally to the spinal cord. The m2 subtype was most abundant in thalamus-hypothalamus and ponsmedulla. The m4 subtype was found in greatest amount in the striatum, whereas m3 and m5 were expressed consistently throughout the CNS. The combination of RT-PCR and HPLC provides a rapid and sensitive method for quantifying the expression of mRNA coding for all five muscarinic receptor subtypes derived from the CNS.

Animals↗
PubMed1998

Prostaglandin E2 alterations during sepsis are partially mediated by endotoxin-induced inhibition of prostaglandin 15-hydroxydehydrogenase.

Prostaglandin E2 (PGE2) is significantly elevated in the plasma of septic or injured patients and is thought to be a component of the resultant immune suppression associated with augmented rates of infection and mortality. Many studies have examined the effect of burn injury and sepsis on PGE2 synthesis. However, the effect of sepsis or burn injury on the expression of prostaglandin 15-hydroxydehydrogenase (PGDH), the key enzyme responsible for PGE2 degradation, has not been explored. The aim of this study was to examine the effect of endotoxin treatment and/or burn injury on the expression of PGDH. Male BDF1 mice were assigned to four groups (n = 4/group): sham, lipopolysaccharide (LPS) (2.5 mg/kg, Escherichia coli LPS, i.p.), burn (15% body surface area scald injury), and burn + LPS (15% body surface area + 2.5 mg/kg LPS, i.p.). Lung tissue was harvested at specific time points after treatment and subsequently was processed for total RNA and protein. Northern and Western blot analyses were used to examine differences in PGDH protein and mRNA expression. Total RNA was probed with the riboprobe for murine PGDH, and the 100,000 g protein fraction was immunoblotted using an rabbit antimurine PGDH antibody. PGDH was expressed in lung at t = 0 in both the saline and LPS-treated animals. A decrease in mRNA expression was initially observed at 2 hours after LPS treatment. The decrease was also significant (p < 0.05) at 3 hours after LPS and maximal decrease in mRNA and protein expression was observed at 6 hours. At 24 hours after LPS administration, the PGDH mRNA and protein expression was still significantly depressed to 49% of control expression. PGDH expression was similar and not statistically different in both burn and burn + LPS treatment at t = 0. At 2 hours after LPS, PGDH mRNA expression in the burn + LPS treatment group had significantly decreased to 47% in comparison with the burn alone group. Maximal decrease in PGDH mRNA and protein expression in lung from burn + LPS was observed at 6 hours after LPS treatment. This change represents a 73% decrease in mRNA in comparison with the time-matched burn control. At 24 hours after LPS administration, PGDH mRNA but not protein expression in the lung from burn + LPS treated mice was still significantly decreased. In summary, LPS treatment alters PGDH mRNA expression at the transcriptional and protein levels. Consequently, sepsis-induced increases in PGE2 levels may not be only due to increased PGE2 synthesis but also due to decreased PGDH expression and, hence, PGE2 degradation.

Animals↗
PubMed1985

Differential stability of host mRNAs in Friend erythroleukemia cells infected with herpes simplex virus type 1.

The consequences of herpes simplex virus type 1 infection on cellular macromolecules were investigated in Friend erythroleukemia cells. The patterns of protein synthesis, examined by polyacrylamide gel electrophoresis, demonstrated that by 4 h postinfection the synthesis of many host proteins, with the exception of histones, was inhibited. Examination of the steady-state level of histone H3 mRNA by molecular hybridization of total RNA to a cloned mouse histone H3 complementary DNA probe demonstrated that the ratio of histone H3 mRNA to total RNA remained unchanged for the first 4 h postinfection. In contrast, the steady-state levels of globin and actin mRNAs decreased progressively at early intervals postinfection. Studies on RNA synthesis in isolated nuclei demonstrated that the transcription of the histone H3 gene was inhibited to approximately the same extent as that of actin gene. We concluded that the stabilization of preexisting histone H3 mRNA was responsible for the persistence of H3 mRNA and histone protein synthesis in herpes simplex virus type 1-infected Friend erythroleukemia cells. The possible mechanisms influencing the differential stability of host mRNAs during the course of productive infection with herpes simplex virus type 1 are discussed.

Animals↗
PubMed2002

Decreased expression of heat shock protein 72 in skeletal muscle of patients with type 2 diabetes correlates with insulin resistance.

Oxidative stress has been ascribed a role in the pathogenesis of diabetes and its complications, and stress proteins have been shown to protect organisms in vitro and in vivo against oxidative stress. To study the putative role of one of the most abundant cytoprotective stress proteins, inducible cytoplasmic 72-kDa-mass heat shock protein (Hsp-72), in the pathogenesis of diabetes, we measured its mRNA concentration in muscle biopsies from six type 2 diabetic patients and six healthy control subjects (protocol 1) as well as in 12 twin pairs discordant for type 2 diabetes and 12 control subjects undergoing a euglycemic-hyperinsulinemic clamp in combination with indirect calorimetry (protocol 2). The amount of Hsp-72 mRNA in muscle was significantly lower in type 2 diabetic patients than in healthy control subjects (in protocol 1: 5.2 +/- 2.2 vs. 53 +/- 32 million copies of Hsp-72 mRNA/microg total RNA, n = 6, P = 0.0039; in protocol 2: 3.2 +/- 3.3 vs. 43 +/- 31 million copies of Hsp-72 mRNA/microg total RNA, n = 12, P = 0.0001). Hsp-72 mRNA levels were also markedly reduced in the nondiabetic co-twins compared with healthy control subjects (5.8 +/- 5.0 vs. 43 +/- 31, n = 12, P = 0.0001), but they were also statistically significantly different from their diabetic co-twins when the difference between the pairs was compared (P = 0.0280). Heat shock protein mRNA content in muscle of examined patients correlated with the rate of glucose uptake and other measures of insulin-stimulated carbohydrate and lipid metabolism. In conclusion, the finding of decreased levels of Hsp-72 mRNA in skeletal muscle of patients with type 2 diabetes and its relationship with insulin resistance raises the question of whether heat shock proteins are involved in the pathogenesis of skeletal muscle insulin resistance in type 2 diabetes.

Blood Glucose↗
PubMed1998

Identification of differentially expressed mRNA in prokaryotic organisms by customized amplification libraries (DECAL): the effect of isoniazid on gene expression in Mycobacterium tuberculosis.

Understanding the effects of the external environment on bacterial gene expression can provide valuable insights into an array of cellular mechanisms including pathogenesis, drug resistance, and, in the case of Mycobacterium tuberculosis, latency. Because of the absence of poly(A)+ mRNA in prokaryotic organisms, studies of differential gene expression currently must be performed either with large amounts of total RNA or rely on amplification techniques that can alter the proportional representation of individual mRNA sequences. We have developed an approach to study differences in bacterial mRNA expression that enables amplification by the PCR of a complex mixture of cDNA sequences in a reproducible manner that obviates the confounding effects of selected highly expressed sequences, e.g., ribosomal RNA. Differential expression using customized amplification libraries (DECAL) uses a library of amplifiable genomic sequences to convert total cellular RNA into an amplified probe for gene expression screens. DECAL can detect 4-fold differences in the mRNA levels of rare sequences and can be performed on as little as 10 ng of total RNA. DECAL was used to investigate the in vitro effect of the antibiotic isoniazid on M. tuberculosis, and three previously uncharacterized isoniazid-induced genes, iniA, iniB, and iniC, were identified. The iniB gene has homology to cell wall proteins, and iniA contains a phosphopantetheine attachment site motif suggestive of an acyl carrier protein. The iniA gene is also induced by the antibiotic ethambutol, an agent that inhibits cell wall biosynthesis by a mechanism that is distinct from isoniazid. The DECAL method offers a powerful new tool for the study of differential gene expression.

Base Sequence↗
PubMed1978

[Effect of prednisolone on the nucleic acid content and RNA synthesis in human lymph nodes in systemic diseases].

Studies on quantitative DNA content in lymphnode cells in patients with lymphogranulomatosis and reticulosis indicated that small prednisolon concentrations (0.1 mg/100 mg of cells) may be responsible for a reduced amount of DNA. The study of the quantitative content and turnover rate of total RNA has shown by the labeled uridine-H3 precursor incorporation a considerable decrease of RNA content and the reduce incorporation of the label in total RNA (approximately by 40% on an average).

Clinical Trials as Topic↗
PubMed1986

Synthesis of mitochondrial macromolecules in herpes simplex type 1 virus infected Vero cells.

How viral infections affect host cell mitochondrial functions is largely unknown. In this study, uptake of radiolabeled precursors was used to assess how a herpes simplex virus type 1 (HSV 1) infection influences synthesis of macromolecules comprising Vero cell mitochondria. Total macromolecular synthesis in infected cells was determined for comparative purposes. Mitochondrial and total cellular DNA syntheses were approximately halved at 1-2.5 h postinfection (PI). Mitochondrial DNA synthesis in infected cells then rose to 3.5-fold that in control cells at 3-4.5 h PI. Total DNA synthesis in infected cells also rose, but more slowly, reaching threefold that for control cells at 5-6.5 h PI. Mitochondrial and total RNA synthesis in infected cells were both decreased by approximately 40% at 1-3 h PI. Over the next 4 h, total RNA synthesis in infected cells slowly continued to decrease, while that in mitochondria recovered to control levels. Synthesis of mitochondrial proteins in infected cells decreased progressively, dropping to about 60% of control levels by 5-6.5 h PI. With the metabolic inhibitors ethidium bromide and cycloheximide, it was determined that nuclear DNA and mitochondrial DNA and mitochondrial DNA directed synthesis of mitochondrial proteins were each partially inhibited in infected cells. Total cellular protein synthesis was decreased by 30% at 1-2.5 h PI and then recovered to control levels by 5-6.5 h PI. Finally, phospholipid synthesis in mitochondria from infected cells was elevated 2.3-fold at 1-5 h PI, but dropped to 14% below control levels during 4-8 h PI.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2007

Real-time RT-PCR (TaqMan) assays for the detection of Grapevine Leafroll associated viruses 1-5 and 9.

Real-time RT-PCR (TaqMan) assays were developed for the specific detection of Grapevine Leafroll associated viruses 1-5 and 9 (GLRaV-1-5 and -9). The assays were evaluated against a wide range of geographically distributed isolates. Geographical locations included South Africa, Europe, Australia, Asia, Latin America and the United States. Sequences were piled up from the most conserved regions of these geographically diverse isolates and TaqMan primers and probes were designed, targeting the regions with 100% sequence identity. Improving the RNA extraction technique and testing the quality of the RNA using the 18S ribosomal RNA TaqMan assay as an RNA specific internal control to validate the quality of the extracted RNA proved to generate better diagnostic assays. The real-time TaqMan RT-PCR assays were compared to the conventional RT-PCR assays for the detection of viruses using purified total RNA as well as crude extract. The data showed that when using total RNA extracted either by the Qiagen RNeasy method or by an ABI automated system more isolates were detected in comparison to crude extract. The optimum volume of crude extract prepared in GES for use in real-time TaqMan RT-PCR cocktail was determined to be 1 microl per reaction. In addition this report showed that TaqMan RT-PCR was more sensitive than conventional one-step RT-PCR for testing different isolates of these viruses either using RNA or crude tissue extract.

Closteroviridae↗
PubMed1987

Detection of estrogen receptor mRNA in human uterus.

We transcribed a cDNA clone of the human estrogen receptor (ER) with T7 RNA polymerase. The 32P-cRNA transcript complementary to ER mRNA was hybridized to poly(A)+ RNA from human uterus and revealed a single band of approximately 4.2 kilobases. No hybridization was seen with the cRNA probe of the opposite orientation. Hybridization of total RNA from calf and rat uterus yielded a single band at approximately 3.8 kilobases for both species. Total RNA from rat spleen did not hybridize. A 35S-labeled cRNA probe was prepared for in situ hybridization of ER mRNA in human uterus and spleen. Autoradiographic signal was present over endometrial epithelium, stromal cells of the lamina propria, and smooth muscle cells of the myometrium but was absent from sections of spleen. The ER mRNA hybridization label was located over cytoplasm and nuclei of uterine target cells.

DNA↗
PubMed2003

Impact of RNA extraction from limited samples on microarray results.

To move microarray technology into the diagnostic realm, the impact of technical parameters, such as sample preparation and RNA extraction, needs to be understood and minimized. We evaluated the impact of two RNA extraction methods, DNase treatment and the amount of hybridized cDNA probe, on the outcome of microarray results. The results for both RNA extraction methods were comparable, although one method resulted in residual DNA that slightly affected the microarray results. As little as one microgram of total RNA could be used to synthesize a cDNA probe and resulted in a gene expression profile that was similar to one produced using 5 micrograms total RNA, even though the overall signal intensity was lower. These experiments illustrate that microarray technology holds great promise for the use of limited clinical samples in the diagnostic setting.

Artifacts↗
PubMed2002

Molecular biological approaches to neurological disorders including knockout and transgenic mouse models.

Advances of molecular biology have provided a great variety of new approaches to research on human disorders. This article gives an outline of molecular biological approaches to analysis of neurological disorders such as giant cell glioblastoma (GGBM) and amyotrophic lateral sclerosis (ALS), and their respective animal models: p53 knockout mice for GGBM and mutant superoxide dismutase-1 transgenic mice for ALS. Genomic DNA extracted from fresh-frozen tissue is examined by Southern blotting for screening mutations in a certain gene. Polymerase chain reaction (PCR) products of a gene in genomic DNA are examined by single-stranded conformation polymorphism, sequencing and agarose gel electrophoresis for identifying mutations, and for preparing and evaluating DNA probes used in Southern blotting and DNA in situ hybridization (ISH). Total RNA from tissue is examined by northern blotting for quantifying and verifying a certain mRNA. Reverse transcription-PCR products of a certain mRNA in total RNA are examined by sequencing and agarose gel electrophoresis for preparing and evaluating cDNA probes used in northern blotting and mRNA ISH. Tissue total protein is immunoblotted for quantifying and verifying a certain protein, and for evaluating the specificity of antibodies used in western blotting and immunohistochemistry. Immunoprecipitates are immunoblotted for evaluating a profile of protein or other substances. Enzyme-linked immunosorbent assay is used for measuring tissue concentration of protein or other substances, and for determining titers of specific antibodies. By these procedures, chronological analysis of animal models for human diseases contribute to elucidating pathogenic mechanisms and exploiting new therapies. Noticing both the similarity and difference between human and animal disorders will help understand the nature of disease.

Amyotrophic Lateral Sclerosis↗
PubMed1976

Inhibition and recovery of ribosomal RNA synthesis in ultraviolet-irradiation mammalian cells.

The effects of ultravilet light on rRNA synthesis were studied in stationary, monkey kidney CV-1 cells. Separate cultures were irradiated with a single ultraviolet dose of up to 525 erg/mm2 at different time intervals before incubation with [3H]uridine. The labeled RNA prepared from whole cells was analyzed by polyacrylamide-agarose gel electrophoresis. In cells ultraviolet irradiated 30 min before transcription analysis, the rate of total RNA and of 45 S RNA synthesis are depressed whereas a synthesis of unusual RNAs, shorter than 45 S RNA, appears. The rates of 28 S and of 18 S RNA synthesis decrease as a function of ultravilet dose and at the same time the ratio of 18 S to 28 S RNA increases. These results are consistent with the idea of a premature RNA chain termination on ultraviolet-damaged DNA. The ultraviolet-induced alterations of transcription disappear progressively with time after irradiation: the rates of total RNA and of 45 S RNA synthesis increase whereas the synthesis of unusual RNAs decreases; in parallel the rates of 28 S and 18 S RNA synthesis recover with a shift of the 18 S to 28 S RNA ratio toward the normal value 1 : 1, probably as a direct consequence of the repair of ultraviolet-induced lesions in the DNA. The kinetics of recovery are dose dependent and are comparable to those of the excision-repair of DNA photoproducts as measured by unscheduled DNA synthesis in ultraviolet-irradiated CV-1 cells. Fluorodeoxyuridine does not affect recovery. The data also show that in CV-1 cells, rRNA genes are arranged after their promotor in the order 18 S rRNA -- 28 S rRNA.

Cell Line↗
PubMed1993

Human thyroid tissue do not express thyroalbumin.

Thyroid tissue total RNAs from multinodular goiter (G2) and from hereditary goiter with defective Tg synthesis (JNA) were hybridized with a 5'albumin cDNA probe (F-47), a 3' albumin cDNA probe (B-44) and a thyroglobulin cDNA probe (phTgM3). JNA refers to tissue obtained from a patient with virtual absence of Tg in thyroid tissue and the presence of increased concentration of an albumin-like labeled protein in the thyroid. No hybridization signal was detected in both G2 and JNA with albumin probes at Northern Blot studies. Those results were confirmed by dot-blot analysis of total RNA where no hybridization signal was detected in G2 and JNA. To confirm that thyroid tissues do not express thyroalbumin total RNA from JNA and normal control thyroid tissue (C) were amplified by PCR using albumin and Tg primers. An expected fragment of 592 bp was observed in a human liver sample with the albumin primers. However JNA and C samples showed absence of an amplification product of the same size. We concluded that thyroid cells do not contain the albumin transcript. Albumin is probably taken up from circulation and iodinated by the thyroid follicular cell with subsequent release of iodoalbumin into the circulation.

Albumins↗
PubMed1998

Granulocyte-colony stimulating factor inhibits inducible nitric oxide synthase gene expression in pulmonary epithelial cells in vitro.

Excessive release of nitric oxide (NO) is most likely a crucial pathophysiological pathway leading to pulmonary dysfunction. Thus, repression of supranormal NO production might be one beneficial mechanism of granulocyte-colony stimulating factor (G-CSF) in inflammatory processes. The aim of this study was to investigate the influence of G-CSF on inducible nitric oxide synthase gene expression in the alveolar epithelial cell line L2. We show that G-CSF suppresses interferon-gamma/tumor necrosis factor-alpha (TNF-alpha) induced inducible nitric oxide synthase gene expression detected as inducible nitric oxide synthase cDNA (cDNA concentration was 633 +/- 38 amol/microg total RNA following 24 h incubation with 100 U/ml interferon-gamma/500 U/ml TNF-alpha, and 440 +/- 14 amol/microg total RNA following 24 h incubation with 250 U/ml G-CSF +/- 100 U/ml interferon-gamma/500 U/ml TNF-alpha, respectively). In addition, application of G-CSF resulted in a decreased synthesis of inducible nitric oxide synthase protein and diminished NO release mediated by the cytokines. The suppression of inducible nitric oxide synthase gene expression in L2 cells by G-CSF may represent a beneficial counterregulatory effect on excessive NO synthesis induced by proinflammatory cytokines in the lung.

Animals↗
PubMed1991

Human complement factor H. Tissue specificity in the expression of three different mRNA species.

Using cDNA clones H-19 and H-46, we have shown previously that three different mRNA species (4.3 kb, 1.8 kb and 1.4 kb) for complement factor H are expressed constitutively in human liver. Here we report data suggesting that the expression of these different factor-H mRNA species is regulated by tissue-specific control mechanisms. Total RNA and poly(A)-enriched RNA from various human tissues (heart, lung, temporal cortex, kidney, spleen, bone marrow and muscle) various cell lines (HepG2, HepG3, HepG4, Hep3B, H-4, Jurkat, Molt4, H-9, KHos24Os, A-431, U937, Mono Mac 6 and Raji) and from primary cultures of peripheral blood monocytes, fibroblasts and human umbilical vein endothelial cells (HUVEC) were investigated for the expression of factor-H mRNA. In RNA preparations from extrahepatic tissue, factor-H mRNA was only detected in biopsies from the lung. Using 20 micrograms total RNA isolated from all 13 cell lines it was not possible to detect any factor-H mRNA, while mRNA for factor H was expressed in monocytes, HUVEC and fibroblasts. When expressed in extrahepatic tissues, only the 4.3-kb and the 1.8-kb mRNA species were detected, while the 1.4-kb mRNA is expressed abundantly in liver. Interferon-gamma did not induce the expression of factor-H mRNA in any of the cell lines tested. On the other hand, tumour necrosis factor-alpha induced the expression of the 4.3-kb mRNA species in U937 cells. In HUVEC and fibroblasts the relative quantities of the 4.3-kb and the 1.8-kb mRNA species and the regulatory effects of interferon-gamma, interleukin-1, dexamethasone and retinoic acid on their expression showed significant tissue specificity.

Blotting, Northern↗
PubMed1995

Reduced levels of mRNA encoding endoskeletal and growth-associated proteins in sensory ganglia in experimental diabetes.

This study investigated changes in levels of mRNAs encoding the three neurofilament (NF) proteins NF-L (low), NF-M (medium), and NF-H (high) and two growth-associated proteins, GAP-43 and T alpha 1 alpha-tubulin, in lumbar dorsal root ganglia of control and streptozocin-induced diabetic rats. After 8 weeks of diabetes the animals were killed, and total RNA was isolated from the L4 and L5 dorsal root ganglia and subjected to Northern blotting, with constant amounts of total RNA loaded onto each lane. A truncated sense RNA for GAP-43 was included as an internal standard during RNA isolation to enable accurate quantification of mRNA levels. The filters were probed sequentially with 32P-labeled cDNAs encoding NF-L, NF-M, NF-H, GAP-43, T alpha 1 alpha-tubulin, and citrate synthase. Hybridizing RNAs were detected by autoradiography and quantified by image analysis. Hybridization signals were normalized to those of the internal standard. In diabetes, NF-L mRNA levels (2.5- and 4-kilobase [kb] transcripts) were decreased by 35 (P = 0.002) and 34% (P < 0.001), respectively, the NF-H mRNA level was decreased by 65% (P < 0.001), but the NF-M mRNA remained unchanged. T alpha 1 alpha-tubulin and GAP-43 mRNA levels were reduced by 56 (P < 0.001) and 30% (P < 0.05), respectively. Levels of citrate synthase mRNA were unchanged. These data indicate a selective defect of expression of growth-associated and endoskeletal proteins in experimentally induced diabetes.

Animals↗
PubMed2003

Reproducibility of oligonucleotide arrays using small samples.

BACKGROUND: Low RNA yields from small tissue samples can limit the use of oligonucleotide microarrays (Affymetrix GeneChips). Methods using less cRNA for hybridization or amplifying the cRNA have been reported to reduce the number of transcripts detected, but the effect on realistic experiments designed to detect biological differences has not been analyzed. We systematically explore the effects of using different starting amounts of RNA on the ability to detect differential gene expression. RESULTS: The standard Affymetrix protocol can be used starting with only 2 micrograms of total RNA, with results equivalent to the recommended 10 micrograms. Biological variability is much greater than the technical variability introduced by this change. A simple amplification protocol described here can be used for samples as small as 0.1 micrograms of total RNA. This amplification protocol allows detection of a substantial fraction of the significant differences found using the standard protocol, despite an increase in variability and the 5' truncation of the transcripts, which prevents detection of a subset of genes. CONCLUSIONS: Biological differences in a typical experiment are much greater than differences resulting from technical manipulations in labeling and hybridization. The standard protocol works well with 2 micrograms of RNA, and with minor modifications could allow the use of samples as small as 1 micrograms. For smaller amounts of starting material, down to 0.1 micrograms RNA, differential gene expression can still be detected using the single cycle amplification protocol. Comparisons of groups of four arrays detect many more significant differences than comparisons of three arrays.

Nucleic Acid Hybridization↗
PubMed2000

Detection of chemical-induced differential expression of rat hepatic cytochrome P450 mRNA transcripts using branched DNA signal amplification technology.

The importance of the cytochrome P450 (CYP) enzyme family in xenobiotic metabolism, as well as their differential expression and activity in response to a wide range of environmental chemicals and pharmaceuticals, is well documented. The objective of this study was to evaluate the specificity of the branched DNA (bDNA) signal amplification technique for the detection of multiple rat CYPs from hepatocellular RNA. Oligonucleotide probe sets were designed to various chemically inducible rat CYP mRNA transcripts, including CYP1A1, CYP1A2, CYP2B1/2, CYP2E1, CYP3A1/23, and CYP4A2/3. The robustness of the bDNA assay was assessed with the CYP2B1/2-specific probe set, and total RNA was isolated from control and phenobarbital (PB)-treated rats. Analysis of these RNA samples by bDNA signal amplification resulted in a linear quantifiable range of RNA detection that spanned three orders of magnitude (0.1-100 microg of total RNA). The fidelity of the bDNA assay was evaluated within a single assay and between assays where repeated measurements of a single sample were reproduced reliably. The specificity of individual CYP probe sets was evaluated with five typical CYP-inducing chemicals on the expression of specific hepatic CYP mRNA transcripts. Male Sprague-Dawley rats were administered 3-methylcholanthrene, PB, isoniazid, pregnenolone-16alpha-carbonitrile, or clofibric acid to induce transcription of CYP1A1, CYP1A2, CYP2B1/2, CYP2E1, CYP3A1/23, and CYP4A2/3 mRNA, respectively. Analysis of chemical-induced differences in gene expression by bDNA signal amplification indicated that 3-methylcholanthrene induced CYP1A1 and CYP1A2 mRNA levels 670- and 11-fold, respectively; PB induced CYP2B1/2 expression 71-fold; pregnenolone-16alpha-carbonitrile induced CYP3A1/23 expression 34-fold; and clofibric acid induced CYP4A2/3 expression 4.7-fold. Overall, these data support the use of bDNA signal amplification technology as a robust, reproducible, and efficient means of monitoring the differential expression of multiple isoforms of the CYP enzyme family.

Animals↗