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At least 325 records · Page 18Linked to original sources
PubMed1999

Accumulation and localisation of maternal RNA in oocytes of Japanese quail.

Accumulation of total RNA and poly(A+)RNA was determined in the oocytes of Japanese quail (Coturnix coturnix japonica) during oogenesis, by a standard spectrophotometric method, after RNA extraction. Intensive RNA accumulation was observed in the oocytes 0.25-2.0 mm in diameter (correlated with the presence of lampbrush chromosomes), followed by a plateau in 2.0-3.0 mm oocytes (correlated with the condensation of lampbrush chromosomes). Resumption of the RNA accumulation in the 3.0-5.0 mm oocytes is interpreted as a result of external RNA delivery by the granulosa cells. Most of the RNA (approximately 3/4, 3-4 micrograms) contained in the ovulated oocyte was found under the vitelline membrane surrounding the yolk. This RNA has been designated as 'extraembryonic RNA', as it is located outside the germinal disc region where the embryo is formed. The extraembryonic RNA is rapidly degraded within 24 h, from ovulation until oviposition, during egg passage through the oviduct, while the RNA present in a germinal disc (approximately 1.1 micrograms) is stable. In bird oocytes the presence of two, territorially separated pools of RNA has been postulated: one contained in a germinal disc and needed for early embryo development, and the second present in the cytoplasmic layer around the yolk supporting oocyte growth and development during oogenesis.

Animals↗
PubMed2003

In vitro binding of hepatitis C virus to CD81-positive and -negative human cell lines.

AIM: The aim of the present study is to study the mechanism of entry of hepatitis C virus (HCV) into human cells. We examined the in vitro binding of HCV to various human cell lines with or without CD81 expression. METHODS: We first used three cell lines, two hepatocyte-derived, huH-7 and HepG2, and one colon cancer cell line, Cw2. Among them, HepG2 did not express TAPA-1 (CD81) on their surface but two others did. They were incubated with HCV + serum for 1 h and HCV-RNA in extracted RNA obtained from these cells was examined by using both a quantitative test and reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: We found that a significant amount of HCV-RNA was detected in huH-7 and HepG2 but not in Cw2. In addition, the titer of HCV-RNA in serum-incubated CD81-transfected HepG2 was similar to that of the non-transfected titer, and the binding between HCV and huH-7 was not inhibited by anti-CD81. Under the same condition, HCV-RNA tended to be detectable in serum-pulsed hepatocyte-derived cell lines, but not in the others. CONCLUSION: These results suggest that while CD81, as reported, specifically binds to HCV-E2 protein, the entry of HCV into human hepatocytes might be regulated by CD81-unrelated molecule(s).

Antibodies↗
PubMed1981

Thyroid hormone attenuates and augments hepatic gene expression at a pretranslational level.

We have attempted to ascertain the proportion of the rat hepatic genome that is under the selective influence of thyroid hormones and to describe the response patterns of individual mRNA sequences in the transition between hypothyroidism and euthyroidism and between euthyroidism and hyperthyroidism. Poly(A)+RNA was extracted from livers of thyroidectomized, intact, euthyroid rats and of thyroidectomized rats rendered euthyroid and hyperthyroid with daily doses of triiodothyronine. The extracted RNA was translated in a reticulocyte lysate system in the presence of [35S]methionine, and the products were analyzed by two-dimensional gel electrophoresis. Triiodothyronine attenuates as well as augments the expression of certain genes at a pretranslational level. This could represent either a direct or an indirect action of the hormone. Triiodothyronine influences approximately 8% of the 231 mRNA sequences visualized, stimulating activity in 11 and inhibiting activity in 7 sequences. Translational activity of at least one mRNA sequence decreased in both thyroidectomized and hyperthyroid animals, compared to euthyroid levels. The relationship of mRNA response to receptor occupancy varied with examples of linear and amplified responses and responses that were maximal at less than full nuclear occupancy.

Animals↗
PubMed2003

Effect of NASA light-emitting diode irradiation on molecular changes for wound healing in diabetic mice.

OBJECTIVE: The purpose of this study was to assess the changes in gene expression of near-infrared light therapy in a model of impaired wound healing. BACKGROUND DATA: Light-Emitting Diodes (LED), originally developed for NASA plant growth experiments in space, show promise for delivering light deep into tissues of the body to promote wound healing and human tissue growth. In this paper we present the effects of LED treatment on wounds in a genetically diabetic mouse model. MATERIALS AND METHODS: Polyvinyl acetal (PVA) sponges were subcutaneously implanted in the dorsum of BKS.Cg-m +/+ Lepr(db) mice. LED treatments were given once daily, and at the sacrifice day, the sponges, incision line and skin over the sponges were harvested and used for RNA extraction. The RNA was subsequently analyzed by cDNA array. RESULTS: Our studies have revealed certain tissue regenerating genes that were significantly upregulated upon LED treatment when compared to the untreated sample. Integrins, laminin, gap junction proteins, and kinesin superfamily motor proteins are some of the genes involved during regeneration process. These are some of the genes that were identified upon gene array experiments with RNA isolated from sponges from the wound site in mouse with LED treatment. CONCLUSION: We believe that the use of NASA light-emitting diodes (LED) for light therapy will greatly enhance the natural wound healing process, and more quickly return the patient to a preinjury/illness level of activity. This work is supported and managed through the Defense Advanced Research Projects Agency (DARPA) and NASA Marshall Space Flight Center-SBIR Program.

Animals↗
PubMed1998

Expression of cellular adhesion regulatory molecule in hepatocellular carcinoma.

Cellular adhesion regulatory molecule (CMAR) enhances the adhesiveness of cells to collagen and laminin and is considered to be a candidate anti-oncogene. The purpose of the present study was to investigate the relationship between the expression of CMAR and clinical features of hepatocellular carcinoma (HCC). Small amounts of liver tissue were obtained from HCC and non-cancerous portions of the liver in 29 patients and from normal liver in seven patients with metastatic liver tumour by biopsy under ultrasound guidance. RNA was extracted with acid guanidinium thiocyanate-phenol-chloroform. Expression of CMAR was assessed by quantitative PCR using beta-actin as an internal standard. A 4 b.p. insertion polymorphism at nucleotide 241 of the CMAR coding region was then investigated using extracted RNA to assess the relationship between the expression of variant mRNA of CMAR and HCC carcinogenesis. The relative expression of CMAR was significantly reduced in HCC compared with non-cancerous and normal livers and had a relationship with certain clinical background factors. The reduced expression of CMAR was thought to be closely associated with the progression of HCC. However, the 4 b.p. insertion polymorphism pattern of CMAR was the same between HCC and non-cancerous liver in all cases in which it was found. These results suggest that progression of HCC may be predicted based on the relative expression of CMAR.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1999

A sensitive nested reverse transcriptase PCR assay to detect viable cells of the fish pathogen Renibacterium salmoninarum in Atlantic salmon (Salmo salar L.).

A nested reverse transcriptase (RT) PCR assay detected mRNA of the salmonid pathogen Renibacterium salmoninarum in samples of RNA extracts of between 1 and 10 cells. Total RNA was extracted from cultured bacteria, Atlantic salmon (Salmo salar L.) kidney tissue and ovarian fluid seeded with the pathogen, and kidney tissue from both experimentally challenged and commercially raised fish. Following DNase treatment, extracted RNA was amplified by both RT PCR and PCR by using primers specific for the gene encoding the major protein antigen of R. salmoninarum. A 349-bp amplicon was detected by polyacrylamide gel electrophoresis and silver stain. Inactivation of cultured bacteria by rifampin or erythromycin produced a loss of nested RT PCR mRNA detection corresponding to a loss of bacterial cell viability determined from plate counts but no loss of DNA detection by PCR. In subclinically diseased fish, nested RT PCR identified similar levels of infected fish as determined by viable pathogen culture. Higher percentages of fish testing positive were generated by PCR, particularly in samples from fish previously subjected to antibiotic chemotherapy where 93% were PCR positive, but only 7% were nested RT PCR and culture positive. PCR can generate false-positive data from amplification of target DNA from nonviable pathogen cells. Therefore, nested RT PCR may prove useful for monitoring cultured Atlantic salmon for the presence of viable R. salmoninarum within a useful time frame, particularly samples from broodstock where antibiotic chemotherapy is used prior to spawning to reduce vertical pathogen transmission.

Animals↗
PubMed1991

Characterization of the heat shock response in Mycobacterium bovis BCG.

We have for the first time characterized the heat shock response in mycobacteria both at the level of transcription, by RNA extraction, Northern (RNA) blotting, and hybridization with gene-specific probes for the Mycobacterium tuberculosis 65- and 71-kDa heat shock proteins (HSPs), and at the level of translation, by [35S]methionine labelling, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography. We observed increased synthesis of 40-, 65-, 71-, and 90-kDa proteins, which appear to be major HSPs in mycobacteria. The 40-, 71-, and 90-kDa HSPs are coordinately regulated in terms of temperature requirements and kinetics of induction but differ in the levels of expression. The 65- and 71-kDa HSPs are differentially regulated in response to temperature, with different kinetics and levels of induction. mRNA transcript sizes for the 71-, 65-, 40-, and 30-kDa proteins were found to be broadly consistent with DNA sequence open reading frames. A maximum increase of about 69-fold in the levels of mRNA for the 71-kDa HSP after 45 min of heat shock at 45 degrees C was observed, whereas the 65-kDa HSP mRNA increased only 5-fold. It was also found that in M. bovis BCG, as in Escherichia coli, a major control mechanism of the heat shock response is operative at the level of transcription. An ability to characterize the heat shock response in mycobacteria provides an experimental model with which to study environmentally regulated gene expression and an opportunity to identify virulence genes, which may coregulate as part of the heat shock regulon.

Blotting, Northern↗
PubMed2004

Arginine increases growth hormone gene expression in rat pituitary and GH3 cells.

The effect of arginine (Arg) and Ornitargin (OT) [a compound containing the aminoacids Arg, citrulline (Cit) and ornithine (Orn)] administration upon growth hormone (GH) gene expression was studied both in vivo and in vitro (hemipituitaries and GH3 cells) by Northern blot analysis. For in vivo studies, adult male Wistar rats were anesthetized, subjected to i.v. infusion of 200 microl of 150 mM NaCl (control group), Arg (15 or 150 mg) or OT (15 mg of Arg, 1 mg of Cit and 4 mg of Orn) at a rate of 20 microl/min, and killed 50 min thereafter. For the in vitro studies, hemipituitaries or GH3 cells were incubated in 1 ml of appropriate medium containing Arg (15 or 150 mg) or OT (15 mg of Arg, 1 mg of Cit and 4 mg of Orn) for 60 min. The pituitaries of the in vivo and in vitro studies and GH3 cells were subsequently processed for RNA extraction. Total RNA was subjected to electrophoresis in agarose (1%)/formaldehyde gel, transferred to a nylon membrane and subjected to hybridization with a rat GH (32)P-cDNA, and (32)P-18S rRNA probe to correct for the variability in RNA loading. After autoradiography of the membrane, the abundance of GH mRNA and 18S rRNA bands was quantified by densitometry. The in vivo study demonstrated that Arg and OT infusion induced a 2.3-fold increase in GH mRNA expression, which could result from the Arg-mediated inhibition of somatostatin release. In addition, in vitro Arg, but not OT, induced GH gene expression in hemipituitaries and GH3 cells, indicating that the aminoacid can act per se at the pituitary somatotrope level. In conclusion, our data show for the first time that arginine stimulates GH gene expression in parallel to its recognized GH-releasing activity.

Analysis of Variance↗
PubMedDate not supplied

Reverse transcriptase polymerase chain reaction on fine needle aspirates for rapid detection of translocations in synovial sarcoma.

OBJECTIVE: To evaluate the utilization of fine needle aspiration (FNA) biopsy to obtain material for reverse-transcriptase polymerase chain reaction (RT-PCR) in the detection of the t(X;18)(p11.2;q11.2) translocation in synovial sarcomas. STUDY DESIGN: We applied RT-PCR to detection of synovial sarcoma fusion gene transcripts on fine needle aspirates. Five clinical samples were first analyzed: one was a tumor previously diagnosed as malignant hemangiopericytoma, one was a poorly defined tumor, and three were suspected synovial sarcomas. FNA material was transferred directly to the RT-PCR reaction tube without RNA extraction. RESULTS: The t(X;18) translocation could be detected on the limited amount of material that FNA provides. In each of the cases studied the representivity of the tumor samples was confirmed microscopically. CONCLUSION: Our protocol permits analysis directly on representative samples without extraction of RNA. The results imply that RT-PCR offers reliable detection of sarcoma fusion gene transcripts on fine needle aspirates. The procedure, apart from being applicable to outpatients, is rapid and sensitive.

Adolescent↗
PubMed1996

Chemokine expression in human oral keratinocyte cell lines and keratinized mucosa.

Chemoattractant cytokines regulate the immune response within the tissue by recruiting neutrophils and macrophages. These so-called chemokines include a large family of peptide molecules encoded by distinct genes. Their expression is controlled by a variety of microbial and host factors. Among host factors, interleukin-1 (IL-1) is thought to be a key regulator of tissue destruction and mediator of the local immune response. To study its influence on chemokine expression, we used a highly sensitive, semi-quantitative method to assess gene expression at the level of mRNA. RNA was extracted from human oral keratinocyte cell lines after treatment with recombinant human IL-1. To test the method further and possibly establish a chemokine mRNA expression pattern, we also extracted RNA from healthy oral keratinized mucosa. Purified RNA was reverse-transcribed and subsequently amplified in a polymerase chain reaction (RT-PCR) by means of specific primer pairs. Amplified sequences were analyzed by agarose gel electrophoresis, visualized by ethidium bromide staining, transferred to nylon membranes, and hybridized to biotinylated oligonucleotide probes. Detection was achieved by streptavidin-conjugated alkaline phosphatase, a chemiluminescent substrate, and autoradiography. Autoradiographs were analyzed by densitometric measurements. IL-1 stimulation resulted in an increase of the chemokine mRNAs encoding interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), and GRO gamma. Macrophage inflammatory protein-1 alpha (MIP-1 alpha) mRNA was not detectable in keratinocytes. In healthy oral mucosa, we found considerable variation between the subjects. Detection of chemokine mRNAs by RT-PCR proved to be sensitive, specific, and fast. It allows for the study of not only cell-line-derived RNA, but also of RNA isolated directly from biopsy material. The latter feature makes this method well-suited for diagnostic purposes.

Cell Line↗
PubMed1991

Transferrin gene expression and transferrin immunolocalization in developing foetal rat lung.

In previous studies we have shown that transferrin (Tf) specifically stimulates dermatan- and chondroitin-sulphate proteoglycan accumulation around lung cells, and in the extracellular matrix of lung tissue, in vitro. The aim of this study was to determine whether the gene for Tf was activated in specific lung cells during development, and whether the protein product showed evidence of association with extracellular matrix. The expression of the gene in developing lung was shown by the hybridization of a Tf cDNA to a 2.4 kb (kilobase) mRNA species in total RNA extracts of foetal lung. The expression of the Tf gene in comparison to a control gene (GAPD, glyceraldehyde phosphate dehydrogenase) was greatest in 19, 20 and 21 day foetal lung, rising from low levels on day 18 and decreasing markedly at term (day 22). Extracts of RNA from primary cultures of mesenchymal fibroblasts and type II epithelial cells were also analysed for Tf mRNA. These experiments indicated that Tf gene expression was predominantly confined to the mesenchymal compartment. The presence of Tf in histological sections of foetal lung was demonstrated by immunohistochemistry and showed a distinct pattern, with intense staining of the alveolar and the capillary basement membranes. The matrix surrounding the mesenchymal fibroblasts was stained in a diffuse network while epithelial cells were unstained. The staining was low from days 12-16 of gestation, increased to a maximum at days 19-20 but decreased markedly toward term. The Tf staining did not co-localize with transferrin receptor, also demonstrated by immunohistochemistry. These results suggest that Tf is not only present at specific sites in the developing lung, but also is synthesized according to a strict developmental schedule of gene expression.

Animals↗
PubMed2003

Quantitative analysis and prognostic implication of SARS coronavirus RNA in the plasma and serum of patients with severe acute respiratory syndrome.

BACKGROUND: The availability of an early diagnostic tool for severe acute respiratory syndrome (SARS) would have major public health implications. We investigated whether the SARS coronavirus (SARS-CoV) can be detected in serum and plasma samples during the early stages of SARS and studied the potential prognostic implications of such an approach. METHODS: We developed two real-time quantitative reverse transcription-PCR (RT-PCR) assays, one for the polymerase and the other for the nucleocapsid region of the virus genome, for measuring the concentration of SARS-CoV RNA in serum/plasma samples from SARS patients. Plasma samples were obtained from 12 confirmed SARS patients on the day of hospital admission, as well as on days 7 and 14 after fever onset. Serum samples were also obtained from 23 confirmed SARS patients on the day of hospital admission, 11 of whom subsequently required intensive care. Viral RNA was extracted from the plasma/serum samples. The extracted RNA was subjected to analysis by the RT-PCR assays. RESULTS: The RT-PCR system for the polymerase region detected SARS-CoV RNA in 50% of plasma and 78% of serum samples from SARS patients during the first week of illness. The detection rates for plasma dropped to 25% at day 14 after fever onset. The median serum SARS-CoV concentrations in patients who required and did not require intensive care unit admission during the course of hospitalization were 5800 and 140 copies/mL, respectively (Mann-Whitney test, P <0.005). These data were confirmed by the RT-PCR system for the nucleocapsid region, which showed an even higher detection rate of 87%. The correlation between the results obtained by the two RT-PCR systems was high (Pearson correlation analysis, r = 0.998; P <0.001). CONCLUSION: Plasma/serum SARS-CoV quantification represents a potentially useful early diagnostic and prognostic tool for SARS.

Humans↗
PubMed2003

Quantification of mRNA for the vitamin D metabolizing enzymes CYP27B1 and CYP24 and vitamin D receptor in kidney using real-time reverse transcriptase- polymerase chain reaction.

Critical to an understanding of the control of 1,25-dihydroxyvitamin D (1,25D) activity is a molecular appreciation of the regulation of three genes, 25-hydroxyvitamin D-1alpha-hydroxylase (CYP27B1), 25-hydroxyvitamin D-24-hydroxylase (CYP24) and vitamin D receptor (VDR). We now report the sensitivity, reproducibility and accuracy of a real-time reverse transcriptase-polymerase chain reaction protocol (Taqman) for the quantification of mRNA levels for these genes in total RNA extracted from kidney tIssue. The sensitivity of the protocol was at least 150 copies of mRNA per reaction. Reproducibility, expressed as the coefficient of variation, ranged between 14 and 30% at the level of approximately 10(4) copies of mRNA per reaction. Accuracy was estimated at greater than 95% for each of these mRNAs. This protocol allows for the comparison of absolute mRNA levels in extracted total RNA in kidneys from animals fed diets containing different levels of calcium, ranging from 0.05% to 1%. Serum 1,25D levels were decreased when the dietary calcium concentration was increased (P<0.05). The levels of CYP27B1 mRNA were highest in the animals fed the 0.05% calcium diet (P<0.01). Conversely, CYP24 and VDR mRNA levels were highest in the animals fed the 1% calcium diet (P<0.01). Both CYP27B1 and CYP24 mRNA levels were major determinants of serum 1,25D levels when dietary calcium intakes were varied in these adult animals (Multiple R(2)=0.70, P<0.01). No significant relationship was detected between kidney CYP27B1 and serum parathyroid hormone (PTH) suggesting that serum calcium may regulate CYP27B1 mRNA expression directly during normocalcaemia. Low levels of CYP24 mRNA were associated with high PTH levels. These findings suggest that kidney CYP24 activity, possibly regulated by factors such as PTH, acts in concert with kidney CYP27B1 to control serum 1,25D levels.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗
PubMed2004

[Antigenic and genetic characterizations of group A influenza viruses H3N2 circulated in men in China during 2000-2002].

OBJECTIVE: To understand the antigenic and genetic characteristics of influenza A H3N2 viruses circulated in man in China from 2000 to 2002. METHODS: Embryonated chicken eggs inoculated with virus for amplification of viral yield. The harvesting egg allantoic fluids with influenza viruses were provided for testing antigen and RNA extraction. Virion RNA was transcribed into cDNA by reverse transcriptase, cDNA amplified by PCR, and the product of PCR was purified. Afterward RNA sequence analysis was performed by the dideoxynucleotide chain termination method using synthetic oligodeoxynucleotide primers. Finally the phylogenetic tree was analyzed with MegAlign software. RESULTS: The H3N2 viruses isolated during 2000-2002 were different in amino acid sequences on HA1 domain protein molecule from those of A/Wuhan359/1995 H3N2 as well as those of A/Sydney/7/1997 H3N2 strains. There were four different positions of amino acid sequence on HA1 domain protein molecule among the H3N2 viruses isolated in 2000 and during 2001-2002. They located at 83, 186, 202, 222 and 225 position, respectively. Of them 83 and 186 were in antigenic determinant E and B, respectively. The others located at left wall of the receptor binding site (RBS). CONCLUSION: From the end of 2001 to the beginning of 2002, the influenza epidemic in Northern China caused by H3N2 virus was due to occurrence of antigenic and genetic changes of influenza A(H3N2) virus.

Amino Acid Sequence↗
PubMed1982

[The role of immune RNA in the immunotherapy of malignant brain tumor (author's transl)].

At present, nonspecific immunotherapy using BCG or Picibanil is popular as an immunotherapy for malignant brain tumor. On the other hand, specific immunotherapy using lymphocytes which play an important role in the passive transmission of tumor immunity is also performed. But this therapy has some disadvantage such as the problem of blocking factor or histocompatibility. To compensate for this disadvantage, specific immunotherapy using the chemical mediater, immune RNA, extracted from immunized lymphocytes has started. We extracted immune RNA from the spleen of immunized rat using phenol and SDS, and observed the effect of normal lymphocytes preincubated with immune RNA in vitro and in vivo experiments. In in vitro experiment, following results were obtained. (1) Normal lymphocytes incubated with immune RNA revealed a significant cytotoxicity and this effect was disappeared by the pretreatment of immune RNA with RNase. (2) Cytotoxic effect of immune RNA seemed to be a tumor specific response. (3) Not only syngeneic RNA, but also xenogeneic RNA were effective. In addition to these results, we decided the optimal condition in which immune RNA transferred the cell mediated cytotoxicity to normal lymphocytes. Next, we tried the purification of immune RNA by way of sucrose density gradient centrifugation method and oligo (dT) cellulose affinity chromatography method. Active fraction of immune RNA was contained in 8-16 S fraction and in poly (A) containing fraction. As in vivo experiment, we injected immune RNA incubated lymphocytes intravenously to brain tumor-bearing rats and observed for death daily. In this experiments, significant prolongation of mean survival time was recognized in the group treated with immune RNA. We now consider the clinical application of immune RNA for malignant brain tumor by the following method: immune RNA is obtained from the lymphocytes of sheep which has immunized with patient's tumor cells. And then patient's own lymphocytes strengthened by incubation with immune RNA is injected intratumorally by way of Ommaya's reservoir. To do this therapy safely and effectively, further purification of immune RNA and solution of its physiochemical characteristics will be necessary.

Animals↗
PubMed1997

Evaluation of tyrosinase mRNA as a tumor marker in the blood of melanoma patients.

PURPOSE: The value of tyrosinase messenger RNA (mRNA) detection in the peripheral blood by reverse-transcription polymerase chain reaction (RT-PCR) as a melanoma marker remains controversial. The purpose of this study was to compare the sensitivities of two different blood processing techniques for tyrosinase mRNA detection and evaluate its potential clinical value. METHODS: A total of 50 patients with progressive stage IV melanoma was studied. Two blood processing methods were used: RNA extraction from the whole blood and RNA extraction from density gradient-isolated peripheral-blood mononuclear cells (PBMC). The RNA samples were tested with a sensitive nested-primer RT-PCR assay. RT-PCR results were also correlated with serum lactate dehydrogenase (LDH), treatment status, and presence of visceral versus nonvisceral metastases. RESULTS: Thirteen (26%) of the density gradient and five (10%) of the whole blood processed samples were PCR positive (P = .011). Serum LDH levels were found to be significantly higher in PCR-positive PBMC-processed patients (P = .015). There was no significant difference in the detection rates between visceral versus nonvisceral metastases or between prior treatment versus no prior treatment. CONCLUSION: Using a density gradient method to process the blood samples resulted in a higher detection rate of tyrosinase mRNA than extracting the RNA from the whole blood. However, the relatively low sensitivity in patients with disseminated and progressive disease compared with other reports suggests that tyrosinase mRNA may be of limited value in the management of malignant melanoma.

Adult↗
PubMedDate not supplied

Isolation of high-quality RNA from white spruce tissue using a three-stage purification method and subsequent cloning of a transcript from the PR-10 gene family.

Isolation of PinmIII cDNA homologues from white spruce tissues required a rigorous RNA extraction protocol developed following assessment of three previously reported conifer RNA extraction protocols. Total RNA was extracted via several purification steps designed to minimize binding of phenolics to nucleic acids and was then subjected to caesium chloride ultra-centrifugation. This procedure produced consistently high-quality, intact RNA from both needles and roots with spectrophotometric ratios of approximately 2.0 for both 260/280 nm and 260/230 nm. Total RNA was obtained from the roots of cold-hardened white spruce seedlings for cDNA library construction. More than 2 million recombinant phage particles were generated from 5 microg of a poly(A)+RNA fraction, and ca. 1.3 million cDNA particles were amplified for storage. Approximately 500,000 primary recombinant clones were screened with an heterologous PinmIII cDNA sequence yielding a unique clone, picgl, that was very similar to members of the PR10 gene family.

Cloning, Molecular↗