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 451 records · Page 25Linked to original sources
PubMed1983

Degradation of pre-messenger RNA in bovine thyroid slices; effect of thyrotropin.

Bovine thyroid RNA labeled by incubation of slices in the presence of 32P-orthophosphate were fractionated by a two-step procedure. Total RNA were extracted by gel filtration on AcA 22 in the presence of pronase and separated by Sepharose 2B chromatography. A small fraction of heavily-labeled RNA (giant RNA) was obtained in the void volume (peak I); the major fraction of RNA (smaller than 45 S) was retarded on the column (peak II) and had a low specific radioactivity. Labeled and total RNA of peak I and labeled RNA species of peak II had a DNA-like nucleotide composition and were polyadenylated. In contrast, the nucleotide composition of total RNA of peak II was similar to that of ribosomal RNA and had a very low poly (adenylic acid) content. Pulse-chase experiments showed a precursor-product relationship between the two RNA fractions. These data indicate that labeled RNA of peak I and peak II likely correspond to newly-synthetized pre-mRNA and mRNA, respectively. Thyrotropin induced a decrease in the amount of 32P-labeled pre-mRNA and a proportional increase of 32P-labeled mRNA suggesting a stimulatory effect of the hormone on the degradation of pre-mRNA.

Animals↗
PubMed1997

Prostate-specific antigen (protein and mRNA) analysis in the differential diagnosis and staging of prostate cancer.

We analyzed complexed and free prostate-specific antigen (PSA), the free/total PSA and complexed/free PSA ratios, acid phosphatase, and prostatic phosphatase in serum from 36 patients with prostatic carcinoma and from 48 non-neoplastic control patients (20 with prostatitis and 28 with benign prostatic hyperplasia). Receiver-operating characteristic plots showed that serum PSA was the most efficient variable, singly used, in discriminating neoplastic from non-neoplastic patients. At a cut-off value of 10.0 ng/ml, serum PSA had a diagnostic sensitivity of 87% and a diagnostic specificity of 83%. In particular, three patients with prostatic carcinoma and twenty non-neoplastic controls had serum PSA levels of between 4 and 10 ng/ml. The subsequent analysis of the serum free/total PSA ratio, in this subgroup, using a cut-off level of 15%, allowed us to classify correctly all prostatic cancer cases and 18/20 non-neoplastic diseases. We next analyzed PSA mRNA in circulating cells using an improved reverse-transcriptase polymerase chain reaction dot blot procedure, from six patients with prostatic carcinoma with distant metastases, and in seventeen with localized cancer. The analysis had a high sensitivity (up to dilutions 1:10(6) of total RNA from prostatic cancer cells vs total RNA from normal blood cells). The analysis revealed circulating micrometastatic cells in 3/6 (50%) cases of metastatic cancer and in 4/17 cases of localized cancer. To conclude, serum total PSA combined with the free/total PSA ratio is a very efficient algorithm in discriminating neoplastic from non-neoplastic prostatic diseases, while other mRNA species must be analyzed, in addition to PSA mRNA, in circulating cells to increase the efficiency in detecting metastatic prostatic cancer.

Diagnosis, Differential↗
PubMed1996

Enrichment of middle repetitive element Bm-1 transcripts in translationally active RNA fractions of the silkmoth, Bombyx mori.

The Bm-1 repetitive element family represents a group of transcribed repetitive sequences in the genome of the silkmoth Bombyx mori. In the Bm-5 and BmN permanent cell lines studied here, alpha-amanitin inhibition and nuclear 'run-on' experiments demonstrated that approximately 80% of the Bm-1 transcripts are produced by RNA polymerase II. Bm-1 transcripts are dramatically enriched in poly A+ and polysomal RNA fractions compared to total RNA in these two cell lines. In the Bm-5 cell line, from total to poly A+ and polysomal RNA fractions, Bm-1 transcripts are enriched approximately 4 and 2 times, respectively, while in the BmN cell line these same fractions are enriched about 2 and 19 times compared to total RNA. This suggests that the Bm-1 transcripts may be involved in post-transcriptional processes or control of translation. Our data also revealed less size heterogeneity of Bm-1 transcripts in polysomal as compared to nuclear fractions. In the Bm-5 and BmN cell lines, the size of most transcripts containing Bm-1 sequences increases from approximately 1700 nt in the nucleus to 3000 nt in the polysomal fraction, both fractions with RNA much larger than the Bm-1 consensus sequence (250 bp). This raises the possibility that some Bm-1 elements are transcribed as part of larger transcripts containing mRNA by way of 'read-through', and may be involved in post-transcriptional regulation of gene expression as cis and/or trans acting elements.

Animals↗
PubMed1986

Extensive postmortem stability of RNA from rat and human brain.

The postmortem stability of brain RNA was measured in rat and human samples for up to 48 hr. Whole rat brains, cooled at a rate approximating that of human brains awaiting autopsy, were collected at intervals from 0 to 48 hr after death and frozen. These samples were compared with freshly obtained, unfrozen rat brains. Two potentially independent characteristics of these RNA populations were measured: the recovery or yield of RNA/gram tissue (quantity) and the integrity or extent of degradation (quality). Total RNA yields were similar after all postmortem delays. Hybridization of [32P]-labeled cDNA probes to nitrocellulose filter blots of electrophoretically separated rat brain RNA failed to reveal degradation of the specific rat brain mRNAs during the postmortem period. Similarly, in vitro translation of these same rat total RNA samples produced high molecular weight translation products with no differences between long and short postmortem times. Human cerebral cortex RNA was prepared by the same methods as those used for rat brain from a neurosurgical sample and four other donors with postmortem intervals from 7 to 36 hr. Typically, the yield of total RNA from human brain was 40-50% of the yield from rat brain. When analyzed by RNA gel blot hybridization studies, as for rat brain RNA, human cortical RNA appeared slightly degraded. However, the degree of apparent RNA degradation was not related to the postmortem interval. In vitro translation products of human cortical RNA revealed high molecular weight peptides at all postmortem intervals, but slightly less [35S]incorporation into these bands was found at the longer postmortem intervals relative to the shorter times. Together, these results demonstrate an extensive stability of brain RNA that invites aggressive use of molecular genetic techniques for the study of human neurodegenerative diseases.

Animals↗
PubMed1994

Expression of the CYP19 gene and its product aromatase cytochrome P450 in human uterine leiomyoma tissues and cells in culture.

The conversion of C19 steroids to estrogens occurs in a number of tissues and is catalyzed by a specific form of cytochrome P450, namely aromatase cytochrome P450 (P450arom). Previously, conversion of radiolabeled androstenedione to estrone has been demonstrated in uterine leiomyomas. By use of reverse transcription followed by polymerase chain reaction amplification of total RNA together with a rat cRNA as an internal standard, we detected and quantified P450arom transcripts in total RNA isolated from 32 of the 35 leiomyoma (91%) and from 18 of the 24 adjacent myometrial (75%) tissue samples from 26 women. P450arom transcripts were not detectable in myometrial tissues from disease-free uteri (n = 8). P450arom transcript levels in leiomyomas were similar to those in adipose tissue (normalized to total RNA) and were 1.5- to 25-fold higher than those in adjacent myometrial tissues. We did not find any correlation between P450arom transcript levels and leiomyoma size, histopathology, uterine weight, or patient age. In leiomyoma smooth muscle cells in culture (n = 4) and tissue explants (n = 4), aromatase activity was stimulated by dibutyryl cAMP, and this effect was potentiated by a phorbol ester. These increases in aromatase expression were accompanied by comparable increases in the levels of translatable P450arom mRNA. Treatment with dexamethasone or platelet-derived growth factor did not stimulate aromatase expression. Consistently higher levels of aromatase activity and P450arom transcripts were found in the leiomyoma tissues than in smooth muscle cells in culture (2- to 20-fold). Reverse transcription-polymerase chain reaction analysis of untranslated 5'-termini of mRNA species in leiomyomas revealed the use of primarily promoter II (the ovarian-type promoter) for CYP19 gene transcription. Leiomyomas also contain some transcripts with untranslated exon I.4 (previously found in adipose stromal cells and skin fibroblasts). Placental-type promoter-specific 5'-ends were not present in leiomyomas. We conclude that aromatase expression in leiomyomas is regulated by the rate of CYP19 gene transcription, which is, in turn, regulated by the use of tissue-specific promoters. These findings are consistent with the hypothesis that localized estrogen biosynthesis may be of pathological significance in the promotion of leiomyoma growth.

Aromatase↗
PubMed1995

The AMP-activated protein kinase gene is highly expressed in rat skeletal muscle. Alternative splicing and tissue distribution of the mRNA.

The AMP-activated protein kinase (AMPK) phosphorylates, and thereby inactivates, a number of enzymes involved in the regulation of lipid metabolism. We have studied the expression of the AMPK gene in a variety of rat tissues. The gene is transcribed into a message of approximately 9.5 kb as detected by Northern blotting. Highest expression of the AMPK message was found in skeletal muscle, which contained 20 amol/micrograms total RNA as determined by competitive reverse-transcription/polymerase chain reaction (RT-PCR). In liver, kidney, brain, mammary glands, heart and lung, AMPK mRNA levels ranged over 1-4 amol/micrograms total RNA. Adipose tissue contained less than 1 amol/microgram total RNA. A second AMPK mRNA form was detected by RT-PCR that was 142 bases shorter than the functional transcript. This transcript was apparently generated by alternative splicing of a single exon within the 5'-coding region. The shorter of the two messages, which is not translated into AMPK protein, contributed between 35-60% of AMPK mRNA in most tissues, but only 15-20% in skeletal muscle and heart. As a result, functional AMPK mRNA was sevenfold higher in skeletal muscle than in liver, although AMPK activity was much lower. By Western blotting, relatively large amounts of AMPK protein were detected in skeletal muscle compared to liver. AMPK isolated from skeletal muscle was not activated by treatment with AMPK kinase under conditions where liver AMPK was fully activated. A single 63-kDa polypeptide was immunoprecipitated from rat skeletal muscle using anti-peptide IgG against AMPK. In contrast, two additional polypeptides with apparent molecular masses of 38 kDa and 36 kDa co-precipitated with the 63-kDa AMPK protein from rat liver. These results indicate that the muscle enzyme has a different subunit organization compared to the liver enzyme, which may account for its low catalytic activity. Together, our results indicate a physiological role for AMPK in muscle, in addition to its previously described role in lipid metabolism.

AMP-Activated Protein Kinases↗
PubMed2000

Fingerprinting of cytochrome P450 and microsomal epoxide hydrolase gene expression in human blood cells.

To examine the character and variability of human cytochrome P450 (CYP) and microsomal epoxide hydrolase (mEH) gene expression in human blood cells, we used a highly sensitive, quantitative, competitive reverse transcriptase-coupled polymerase chain reaction (QC RT-PCR) assay to assess mRNA profiles for a battery of 8 genes, in peripheral lymphocytes isolated from 10 healthy donors. Of the genes profiled, in lymphocytes CYP2D6 was typically expressed at the highest levels (3.8 x 10(5) molecules/microg total RNA), with CYP2E1 and mEH also maintained at relatively high abundance (1.2 x 10(5) and 1.8 x 10(5) molecules/microg total RNA, respectively). CYP1A1 levels were approximately an order of magnitude lower (3.9 x 10(4) molecules/microg total RNA), followed by CYP2F1 and CYP3A levels that were near the detection limit of the assay. CYP1A2 and CYP2A6/7 mRNAs were not detected in any of the lymphocyte samples. Overall, relatively low levels of inter-individual variation (2- to 6-fold) existed among these endpoint parameters in the subjects tested. To test whether established human blood cell lines were suitable models to assess basal expression and chemical induction responsiveness of these genes, we determined that constitutive CYP and mEH mRNA profiles were essentially conserved across 4 established human blood cell lines, and highly analogous to the basal expression patterns identified in freshly isolated peripheral lymphocytes. mEH protein was detected in all of the cell lines using Western immunoblotting and chemiluminescent visualization, whereas CYP1A1, CYP2D6, CYP2E1 or CYP3A proteins were not detected in these analyses. When blood cell-derived cultures were exposed to the prototypical CYP1A and CYP3A inducers, i.e., beta-naphthoflavone (beta-NF), dexamethasone (DEX) or phenobarbital, generally little or no inductive response was manifested. Thus, the data obtained from this investigation indicate that, although human blood cell lines in general exhibit poor responsiveness to prototypical inducer exposures, the constitutive patterns of CYP and mEH expression in peripheral lymphocytes appear to exhibit relatively low levels of variation among individuals. In addition, these in vivo patterns of expression are well maintained in established cultured blood-cell lines.

Adult↗
PubMed2004

[Amplification, cloning and expression of 3-phosphoglycerate kinase gene from Clonorchis sinensis].

OBJECTIVE: To construct a prokaryotic expression plasmid encoding 3-phosphoglycerate kinase (PGK) gene of Clonorchis sinensis and analyze its expression in E. coli JM109. METHODS: Total RNA was extracted with Trizol. The gene encoding PGK was amplified by RT-PCR from the total RNA, and was cloned into the pGEX-4T-1 vector. The recombinant plasmids pGEX-4T-1-PGK constructed were transferred into JM109, positive recombinants were screened and identified by tool enzyme digestion, PCR technique and sequencing. The recombinant was induced with IPTG to express the target protein. The expression product was characterized by SDS-PAGE. RESULTS: There were three clear bands from extracted total RNA, the PGK gene (1248 bp) was amplified by RT-PCR technique, the sequencing result was consistent with the known sequence. Ten clones were obtained by screening after transferring, six of which were positive recombinants. The positive recombinant was induced to express, and the SDS-PAGE showed the expression products was about Mr 70000. CONCLUSION: The recombination prokaryotic expression vector pGEX-4T-1-PGK has been reconstructed and a fused protein was expressed which contained Sj26 GST.

Animals↗
PubMed1997

Adenosine A3 receptor expression and function in eosinophils.

The A3 adenosine receptor is widely expressed in human tissues with the most abundant expression in the lung and liver, but the predominant cellular localization and functions of this receptor in humans are unknown. Since adenosine influences the activation of circulating and resident inflammatory cells within the lung and leads to exaggerated airway narrowing in individuals with inflammatory airway disorders, we hypothesized that A3 receptor gene expression is localized to inflammatory cells and that gene expression is upregulated in airway inflammation. Lung and airway tissue were obtained at thoracotomy from nonsmoking subjects and subjects with inflammatory airway disorders associated with tobacco smoke or asthma. In situ hybridization identified A3 receptors in mesenchymal cells and eosinophils within the lamina propria of the airways and the adventitia of blood vessels, but not in mast cells. A3 receptor transcripts were highly expressed in peripheral blood eosinophils purified from atopic donors (6.36 +/- 0.60 pg/microg total RNA) in comparison with neutrophils (0.26 +/- 0.06 pg/microg) or mononuclear cells (0.9 +/- 0.15 pg/microg). Mean A3 receptor transcript abundance was greater in lung tissue from subjects with airway inflammation (0.33 +/- 0.04 pg/microg total RNA) than in normal lung (0.24 +/- 0.03 pg/microg total RNA, P = 0.035). The A3 receptor agonist N6-(4-amino-3-iodobenzyl)adenosine dose-dependently inhibited platelet activating factor-induced eosinophil chemotaxis to a maximum of 41%. This inhibitory effect was completely abolished by addition of the A3 receptor selective antagonist 3-(3-iodo-4-aminobenzyl)-8-(4-oxyacetate)phenyl-1-propylxanthine. We conclude that A3 receptors are primarily expressed on eosinophils in human lung, where they mediate inhibition of eosinophil chemotaxis. Specific A3 receptor ligands may be useful agents in the treatment of eosinophil-dependent diseases such as asthma and rhinitis.

Adenosine↗
PubMed2002

Optimization and evaluation of T7 based RNA linear amplification protocols for cDNA microarray analysis.

BACKGROUND: T7 based linear amplification of RNA is used to obtain sufficient antisense RNA for microarray expression profiling. We optimized and systematically evaluated the fidelity and reproducibility of different amplification protocols using total RNA obtained from primary human breast carcinomas and high-density cDNA microarrays. RESULTS: Using an optimized protocol, the average correlation coefficient of gene expression of 11,123 cDNA clones between amplified and unamplified samples is 0.82 (0.85 when a virtual array was created using repeatedly amplified samples to minimize experimental variation). Less than 4% of genes show changes in expression level by 2-fold or greater after amplification compared to unamplified samples. Most changes due to amplification are not systematic both within one tumor sample and between different tumors. Amplification appears to dampen the variation of gene expression for some genes when compared to unamplified poly(A)+ RNA. The reproducibility between repeatedly amplified samples is 0.97 when performed on the same day, but drops to 0.90 when performed weeks apart. The fidelity and reproducibility of amplification is not affected by decreasing the amount of input total RNA in the 0.3-3 micrograms range. Adding template-switching primer, DNA ligase, or column purification of double-stranded cDNA does not improve the fidelity of amplification. The correlation coefficient between amplified and unamplified samples is higher when total RNA is used as template for both experimental and reference RNA amplification. CONCLUSION: T7 based linear amplification reproducibly generates amplified RNA that closely approximates original sample for gene expression profiling using cDNA microarrays.

Journal Article↗
PubMed1994

Expression of mRNA for natriuretic peptide receptor subtypes in bovine kidney.

The localization of mRNA for atrial natriuretic peptide (ANP) receptor subtypes (A, B, C) in the kidney was examined. Quantitative analysis of the ribonuclease protection assay showed that the numbers of type A receptor (ANPRA) mRNA were 6.9 x 10(7) in the glomeruli and 10.4 x 10(7) molecules/micrograms of total RNA in the inner medulla, and that of type C receptor (ANPRC) mRNA was 21.7 x 10(7) molecules/micrograms of total RNA in the glomeruli. The type B receptor (ANPRB) mRNA was present in smaller numbers (4.5-4.9 x 10(6) molecules/micrograms of total RNA) evenly throughout the kidney fractions. In situ hybridization demonstrated both ANPRA and ANPRC mRNA selectively in the glomerular epithelial cells and ANPRA mRNA in the collecting duct cells of the inner medulla. ANPRC was also localized on the foot processes of glomerular epithelial cells by immunohistochemistry using a specific antibody against the receptor. These results indicate that ANPRA is the major biologically active receptor for the ANP family of hormones in the kidney and is present selectively on the glomerular epithelial cells and inner medullary collecting duct cells. These cells are presumed to play a role in the regulation of glomerular filtration rate and sodium excretion induced by the family of ANP.

Animals↗
PubMed2002

Induction of myeloma-specific cytotoxic T cells using dendritic cells transfected with tumor-derived RNA.

Current immunotherapeutic trials for patients with multiple myeloma (MM) focus on the idiotype (Id) as a tumor-specific antigen for active immunization. To bypass the need for the identification of shared MM-associated antigens and the characterization of possible immunogenic T-cell epitopes in a human leukocyte antigen (HLA) type-restricted manner, we focused on myeloma RNA transfection of dendritic cells (DCs). Total RNA encodes the whole antigen content of tumor cells, therefore allowing the transfected DCs to process and present the most relevant peptides and to induce a possible polyclonal cytotoxic T lymphocyte (CTL) response against different immunogenic antigens. We transfected monocyte-derived DCs with total RNA from the myeloma cell lines LP-1 and U266 by electroporation and investigated the potential of these DCs to induce myeloma-specific CTLs. We show that RNA-transfected DCs induce CTLs that lyse the LP-1 and U266 myeloma cells in an antigen-specific and major histocompatibility complex (MHC) class I-restricted manner, as demonstrated by cold-target inhibition and antibody-blocking studies. Interestingly, LP-1-specific CTLs showed no specificity for the idiotype. Consistent with studies demonstrating mucin 1 (MUC1) as a myeloma-associated antigen, we found MUC1 specificity of the CTLs induced with U266-derived RNA. As corresponding epitopes, we tested the described peptides M1.1 and M1.2 and found a striking fine specificity for M1.2, assuming a possible immunodominance of this peptide. This is the first report on the induction of myeloma-specific CTLs by RNA transfection of DCs.

Cytotoxicity Tests, Immunologic↗
PubMed1999

Regulation of dihydropyrimidine dehydrogenase in colorectal cancer.

Dihydropyrimidine dehydrogenase (DPD) is responsible for degradation of the pyrimidines uracil and thymine and the inactivation of the chemotherapeutic agent 5-fluorouracil. DPD activity is highly variable in cancer populations, and this variation may influence the antitumor efficacy of 5-fluorouracil. However, little is known about the regulation of DPD mRNA expression in any tissues. Using a reverse transcription competitive PCR assay, we quantified DPD mRNA levels in 10 matched colorectal tumors and adjacent normal mucosae and 7 colorectal liver metastases and adjacent normal livers. Lower levels of DPD mRNA expression were observed in colorectal tumor compared with adjacent normal colon mucosa (median, 0.01 versus 0.37 amole/microg total RNA, P = 0.02). DPD mRNA expression was also lower in metastases than adjacent normal liver tissue (median, 0.11 versus 1.17 amole/microg total RNA, P = 0.001). DPD mRNA expression was higher in normal liver than normal colonic mucosa (median, 1.17 versus 0.37 amole/microg total RNA, P = 0.02). A significant relationship was observed between DPD mRNA and catalytic activity (r(s) = 0.66, P<0.001). The tumor:normal ratio for DPD mRNA, protein, and activity was relatively stable in liver (0.25, 0.55, and 0.51, respectively) but varied considerably in colon (0.085, 0.9, and 1.25, respectively), consistent with enhanced translation of DPD transcript in primary colorectal tumor. This suggests that DPD can be regulated at the levels of both transcription and translation.

Colon↗
PubMed1994

A reverse transcription-polymerase chain reaction study of p75 nerve growth factor receptor gene expression in developing rat cerebellum.

The actions of the neurotrophins are mediated through specific receptors. Nerve growth factor (NGF), the prototypic neurotrophin, binds to receptors of both high and low affinity. A protein 75 kDa in size (p75NGFR) binds NGF, as well as brain-derived neurotrophic factor and neurotrophin 3, with low affinity. Recent investigations suggest that this protein may also be a component of the high affinity NGF receptor complex. To study gene expression of the p75NGFR molecule, we used a sensitive reverse transcription-polymerase chain reaction (RT-PCR) assay to measure levels of its messenger RNA (mRNA) in small samples of total RNA. The assay is based on using a shortened p75NGFR cRNA as an internal RNA standard to control for variability in reverse transcription and polymerase chain amplification. We measured p75NGFR mRNA levels in the rat cerebellum during ontogeny to further study the transient developmental increase in receptor gene expression known to occur in this brain region during the early postnatal period. We found that p75NGFR mRNA levels were most abundant at postnatal day 2, and then declined to lower levels throughout postnatal development and in the adult. Northern blot analysis of the same total RNA samples used in our RT-PCR assay verified that p75NGFR expression is highest in the early postnatal period. These results confirm those of previous studies accomplished with much larger amounts of RNA using ribonuclease protection or northern blot assays. The use of an RT-PCR assay that utilized an internal standard also controls against changes in RNA complexity which can affect the measurement of message abundance across developmental stages.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1977

The effects of various kinds of sparsely-ionizing radiation on total cell RNA and preribosomal nuclear RNA of Novikoff hepatoma ascites cells after in vivo labelling.

The effects of 300 kV X-rays, 60Co gamma-rays, 43 MV X-rays and 43 MeV electrons on total cell RNA and 45 S pre-r RNA were investigated in Novikoff Hepatoma ascites cells. Six hours after local irradiation of the animals with 600 rad, the tumour cells were labelled in vivo for 30 and 60 min. The specific activities of high-molecular-weight RNA were influenced differently. This may be due to divergent effects of the applied radiation qualities at the level of transcription and transformation (processing). In addition, these radiation effects are mediated by the adrenal glands in a rather complex manner. Therefore, the results presented in this study support the suggestion that an in vivo labelling system is unsuitable for the evaluation of quantitative radiation effects on RNA.

Animals↗
PubMed1996

Platelet-derived growth factor-A mRNA expression in fetal, normal adult, and atherosclerotic human aortas. Analysis by competitive polymerase chain reaction.

BACKGROUND: To understand which growth factors are important for growth of atherosclerotic plaques, it is necessary to know the factor's relative abundance and how its gene is regulated in relation to cell proliferation. We tested whether platelet-derived growth factor-A (PDGF-A) mRNA levels correlated with cell proliferation in developing aorta, normal adult aorta, and atherosclerotic plaques. METHODS AND RESULTS: We developed a competitive reverse transcription-polymerase chain reaction (RT-PCR) assay to measure human PDGF-A mRNA levels in small tissue samples. A mutated PDGF-A synthetic RNA was used as an internal standard to compete with endogenous PDGF-A mRNA for amplification. The assay is highly sensitive and much more precise than routine RT-PCR. Correction for heteroduplex pairing between the endogenous and mutant PCR products correlates precisely with synthetic RNA standards and quantitative Northern blotting. Immunostaining with the proliferation marker (proliferating cell nuclear antigen) showed the following rank order of proliferation: fetal aorta >> atherosclerotic plaque > normal aortic media. PDGF-A mRNA levels, however, did not correlate with proliferation. Normal adult aorta contained the most PDGF-A mRNa (34.0+/-7.6 amol/microgram total RNA). Fetal aortas were intermediate (10.2+/-1.6 amol/microgram total RNA); advanced atherosclerotic plaques contained the least PDGF-A mRNA (0.3+/-0.1 amol/microgram total RNA). PDGF-A protein was readily detectable in normal media by immunostaining. Advanced plaques generally had less cell-associated PDGF-A protein, although A-chain was also detected in plaque matrix. CONCLUSIONS: PDGF-A mRNA and protein do not correlate with proliferation among these three groups. The significance of high levels of PDGF-A mRNA in the "quiescent" aortic media is unknown but it clearly does not promote cell replication.

Adult↗
PubMed1987

Evidence that expression of asialo-GM1 may be associated with cell activation. Correlation of asialo-GM1 expression with increased total cellular RNA and protein content in normal thymocyte and spleen cell populations.

The question of the biologic significance of asialo-GM1 (aGM1) expression by a limited number of cells including natural killer cells has been raised by the recent demonstrations that aGM1 is expressed by activated macrophages and activated T cells as well as the proliferating thymoblast and functionally mature subpopulations of thymus. The current report demonstrates that the expression of aGM1 on aGM1-negative lymphocytes can be induced by stimulation with mitogens under activating conditions. In addition, activation of the aGM1-negative tumor lines EL-4/F and P388D1/B1 under conditions that result in interleukin 2 or interleukin 1 production, respectively, also result in a significant increase in aGM1 expression by the tumor cell lines. Expression of aGM1 therefore appears to be associated with events occurring as a prelude to or during activation of effector function. Since the aGM1-negative cells do display sialylated versions of aGM1 which can be converted by neuraminidase treatment into serologically recognizable aGM1, it is suggested that the expression of aGM1 might reflect a change in the level of glycolipid sialylation rather than a change in membrane lipid composition per se. The small percentage of lymphocytes in normal, nonstimulated spleen and thymus populations which express significant levels of aGM1 were sorted and analyzed for total cellular protein and RNA. Increases in RNA synthesis and in total cellular protein and RNA above basal levels of resting lymphocytes are considered indicators of a G0----G1 transition. The aGM1-positive populations displayed a larger mean population size (as indicated both by higher forward light scatter and by higher total cellular protein content), and a higher mean population RNA content than the aGM1-negative populations. These data are discussed in the context of the hypothesis that the expression of aGM1 may be associated with early events in the activation of resting cells which prepare the cells for subsequent induction of effector function.

Animals↗
PubMed1991

[Detection of gonadotropin releasing hormone (GnRH) messenger RNA in the ovary and cloning of LH, chorionic gonadotropin (CG) receptor].

Detection of gonadotropin releasing hormone (GnRH) mRNA in rat ovary was carried out, and cloning of LH, chorionic gonadotropin (CG) receptor in human ovary was attempted by use of polymerase chain reaction (PCR). Firstly, in an attempt to detect the expression of GnRH or related gene in rat ovaries at the RNA level, GnRH message was amplified. Total RNA from rat ovaries was converted to cDNA using reverse transcriptase and amplified in PCR using a pair of specific primers complementary to the rat GnRH cDNA. The DNA products were subcloned into plasmid vectors and their sequence determined. 1. In the rat ovary, a prominent PCR product of 462 bp was identified as a fragment of prothymosin alpha cDNA previously found in the spleen. 2. In contrast, RT-PCR amplification of hypothalamus and granulosa cell messages indicated the presence of a 244 bp product with identical sequence to GnRH. To confirm the presence of GnRH message, a second set of GnRH primers was used. PCR amplification of cDNA from hypothalamus, granulosa cells and whole ovary yielded a product identical with the authentic GnRH cDNA sequence. These data demonstrated the presence of mRNA for GmRH and prothymosin alpha in the rat ovary. Secondly, a part of the human LH, CG receptor was obtained from human granulosa cells by selective amplification with PCR of DNA segments presenting possible sequence similarity with genes for the porcine or rat LH, CG receptor. Total RNA from human granulosa cells was converted to cDNA and amplified in PCR using degenerate oligonucleotide primers corresponding to possible conserved regions in extracellular segments of the porcine or rat LH, CG receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗