Reverse Transcription And The Polymerase Chain Reaction Research Articles

Cytokine production suppression by culture supernatant of B16F10 cells and amelioration by Ganoderma lucidum polysaccharides in activated lymphocytes.

Some cytokines, such as interleukin-2 (IL-2), interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), produced by lymphocytes might play an important role in anti-tumor immunity and their production is possibly suppressed by cancer. Amelioration of the suppression of cytokine production might contribute to cancer control. Ganoderma lucidum polysaccharides (Gl-PS), a versatile group of a component of G. lucidum and one with various bioactivities, might have this potential. In this study, analyses including reverse transcription and the polymerase chain reaction (RT-PCR), immunocytochemistry and Western blot were used to test the effects of Gl-PS on the production of IL-2, IFN-γ and TNF-α in mononuclear lymphocytes by incubating Gl-PS with mouse splenic mononuclear lymphocytes in the presence of B16F10 cell culture supernatant following activation by phytohemagglutinin. The RT-PCR, immunocytochemistry and Western blot assays showed that the production of IL-2, IFN-γ and TNF-α in mononuclear lymphocytes was suppressed by B16F10 cell culture supernatant at both the mRNA and protein levels, whereas the suppression was fully or partially ameliorated by Gl-PS. The amelioration by Gl-PS against the suppression of the production of IL-2, IFN-γ and TNF-α in mononuclear lymphocytes by B16F10 cell culture supernatant might contribute to cancer control.

Changes in Estrogen Receptor ERα and ERβ Expression in Chicken (Gallus domesticus) Adrenal Gland during Short-fasting and Refeeding

Estrogen receptors have been found in the adrenal gland of rodents, monkeys, mares and sheep, indicating a connection between sex steroids and the activity of the adrenal gland. In the present study, the expression of estrogen receptors alpha (ERalpha) and beta (ERbeta) in the chicken adrenal gland during stress induced by 24 h fasting and after refeeding was determined using reverse transcription and the polymerase chain reaction (RT-PCR). The presence of both ER mRNAs in the adrenal gland of all examined groups was found. The relative expression of ERalpha mRNA was higher than ERbeta mRNA. There were no significant differences in ERalpha mRNA expression among the examined groups. On the contrary, we observed changes in ERbeta expression during stress conditions. These findings indicate different pathways of estrogen action in the avian adrenal gland. Furthermore, changes in ERbeta level suggest that this form of estrogen receptor plays a predominant role for estrogen action in the chicken adrenal gland during stress.

In situ proliferation and differentiation of macrophages in dental pulp.

The presence of macrophages in dental pulp is well known. However, whether these macrophages proliferate and differentiate in the dental pulp in situ, or whether they constantly migrate from the blood stream into the dental pulp remains unknown. We have examined and compared the development of dental pulp macrophages in an organ culture system with in vivo tooth organs to clarify the developmental mechanism of these macrophages. The first mandibular molar tooth organs from ICR mice aged between 16 days of gestation (E16) to 5 days postnatally were used for in vivo experiments. Those from E16 were cultured for up to 14 days with or without 10% fetal bovine serum. Dental pulp tissues were analyzed with immunohistochemistry to detect the macrophages and with reverse transcription and the polymerase chain reaction (RT-PCR) for the detection of factors related to macrophage development. The growth curves for the in vivo and in vitro cultured cells revealed similar numbers of F4/80-positive macrophages in the dental pulp. RT-PCR analysis indicated the constant expression of myeloid colony-stimulating factor (M-CSF) in both in-vivo- and in-vitro-cultured dental pulp tissues. Anti-M-CSF antibodies significantly inhibited the increase in the number of macrophages in the dental pulp. These results suggest that (1) most of the dental pulp macrophages proliferate and differentiate in the dental pulp without a supply of precursor cells from the blood stream, (2) M-CSF might be a candidate molecule for dental pulp macrophage development, and (3) serum factors might not directly affect the development of macrophages.

Effects of injection of biogenic amines on expression of gill related ion transporter mRNA and α-subunit protein in Litopenaeus vannamei

We used semi-quantitative reverse transcription and the polymerase chain reaction (RT-PCR) to identify and characterize the mRNA of Na +/K +-ATPase α-subunit and Vacuolar-type H/-ATPase (V-ATPase) β-subunit prepared from gills of the white shrimp Litopenaeus vannamei. Western blot and immunocytochemistry analysis were employeed to investigate the effects of biogenic amines such as dopamine (DA) and 5-hydroxytryptamine (5-HT) injection on expression of protein abundance and immunostained sites. Our results show that the gill Na +/K +-ATPase of 5-HT treatments increased lag to those of DA treatments. However, the DA and 5-HT have no significant effects on the activity of V-ATPase. The biogenic amines could induce the synthesis of Na +/K +-ATPase α-subunit mRNA in the first 12 h. The degree of change in Na +/K +-ATPase α-subunit mRNA expression were different between DA and 5-HT treated groups which depend on the dose. The expression of V-ATPase β-subunit mRNA was not markedly affected by the biogenic amines. Concomitant elevation of Na +/K +-ATPase activity, α-subunit mRNA as well as protein abundance in response to injected biogenic amines was also found in Na +/K +-ATPase immunostained sites within the gill epithelium.

Detection of hepatitis E virus in acute hepatitis in Siberia

Samples of blood serum obtained from patients with symptoms of acute hepatitis, admitted to infectious disease hospitals of Novosibirsk, Barnaul, and Irkutsk, were investigated. The serologic markers of hepatitis E virus (HEV): IgM and IgG antibodies, were determined by enzyme immunoassay (ELISA). Seropositive samples were investigated by the method of reverse transcription and the polymerase chain reaction (RTPCR). Two variants of HEV were detected, and the first human cases of HEV infection in this region were demonstrated. One of the variants was identified as genotype I, the other as genotype III of HEV. The HEV variants found were cultured in a continuous culture of green monkey kidney cells (4647). The ability of the HEV variant belonging to genotype I to persist in cell culture was shown.

Expression analysis of genes putatively involved in chicken gonadal development

In mammals, testis development is initiated by the expression of the sex-determining gene, SRY whereas the genetic trigger for sex determination in birds remains unknown. In the present study, the expression of seven genes implicated in vertebrate sex determination and differentiation were studied in chicken embryonic gonads from day 4 to day 12 of incubation using reverse transcription and the polymerase chain reaction (RT-PCR). Results showed transcription of cLhx9, cGATA4, cVnnl, cPptl, cBrd3 were sexually dimorphic during chicken gonadal development, whereas cEki2, cFog2 were expressed at similar levels in both sexes. Results of comparative studies between mammals and chickens show that vertebrate sex-determining pathways comprise both conserved and divergent elements: expression profiles of cGATA4/cFog2 and cVnnl are similar to those in mammals, while others appear some differences. Possible functions of these genes on chicken gonadal development were analyzed based on their expression profiles.

Expression of Tom34 splicing isoforms in mouse testis and knockout of Tom34 in mice.

The 34-kDa translocase of the outer mitochondrial membrane (Tom34) is a putative mammalian-specific factor involved in protein import into mitochondria. We analyzed the genomic sequence of the mouse Tom34 gene and found it has two alternative initial exons. Using reverse transcription and the polymerase chain reaction (RT-PCR), we found that these two mRNAs differs only in the 5'-proximal sequences corresponding to the two initial exons (exon 1a and 1b). Tom34 mRNA with exon 1a (Tom34a) is expressed ubiquitously, while that with exon 1b (Tom34b) is expressed only in mature testicular germ cells. To explore the in vivo function of Tom34 proteins, we generated Tom34-deficient mice by targeted disruption. The Tom34(-/-) mice were viable and grew normally and had a normal Mendelian inheritance pattern. Male as well as female Tom34(-/-) mice were fertile. In vitro-preprotein import into isolated mitochondria showed no apparent difference between Tom34(-/-) and wild-type mice. These results indicate that Tom34 is dispensable for mouse growth and development under optimal conditions.

Expression of genes for alcohol and aldehyde metabolizing enzymes in mouse oocytes and preimplantation embryos

Alcohols and aldehydes are metabolized primarily by alcohol (ADH) and aldehyde (ALDH) dehydrogenase isozymes. Although significant progress has been made towards understanding the involvement of these isozymes in the oxidation of alcohol and aldehydes in the body, it is not known how these compounds are handled during fertilization and preimplantation embryogenesis. In this study, reverse transcription and the polymerase chain reaction (RT-PCR) was used to determine which ADH and ALDH isozymes are expressed at the oocyte, zygote, morula, and blastocyst stages of preimplantation development in the mouse. Transcripts of β-actin and vimentin, assayed as controls, were detected at all stages, as well as Class III ADH ( Adh-2) and Class 3 ALDH ( Ahd-4), involved in the detoxification of formaldehyde and aromatic aldehydes, respectively. In contrast, transcripts for the major ethanol oxidizing isozyme, Class I ADH ( Adh-1) was not detected during preimplantation development. Cytosolic retinol dehydrogenase ( Adh-3) transcripts were marginally detected in oocytes and zygotes. The mRNA for cytosolic retinal dehydrogenase ( Ahd-2), microsomal short-chain retinol dehydrogenases (RoDH Type I), and the mitochondrial low-Km acetaldehyde dehydrogenase ( Ahd-5) only appeared as maternal transcripts. Microsomal ALDH ( Ahd-3), which is induced by 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), was not expressed until the blastocyst stage. ADH and ALDH enzyme systems may guard mouse preimplantation embryos against the toxic effects of industrial pollutants, such as formaldehyde and TCDD, as well as peroxidatic aldehydes generated during lipid peroxidation. The absence of enzymes to convert ethanol to acetaldehyde, coupled with oocyte expression of the acetaldehyde-degrading enzyme, Ahd-5, may be protective for the early embryo.

RT-PCR analyses of differential gene expression in ES-derived neural stem cells.

Reverse transcription and the polymerase chain reaction (RT-PCR) provides a very sensitive method to identify known genes that are both upregulated and downregulated during neuronal differentiation. First strand cDNAs are generated in the initial reaction with reverse transcriptase in the presence oligo-dT primed RNAs. The cDNA provides the template for amplification of gene specific targets with the appropriate primers. The purity of the RNA is extremely important to the success of these procedures. The presence of chromosomal DNA in the RNA preparation will influence the reliability of both RNA concentration and the PCR products generated from these RNA templates in the reverse transcription reactions. Many parameters in PCR have to be optimized for each gene specific primer pair and template; they include primer and MgCl2 concentrations, primer annealing temperatures, and the number of PCR cycles (,).

Gene expression during gonadogenesis in the chicken embryo.

Genes implicated in vertebrate sex determination and differentiation were studied in embryonic chicken gonads using reverse transcription and the polymerase chain reaction (RT-PCR). Expression profiles were obtained during gonadal sex differentiation for AMH, SOX9, SOX3, the Wilm's Tumour gene, WT1, and the orphan nuclear receptor genes, SF1 and DAX1. Some of these genes showed sexually dimorphic expression profiles during gonadal development, whereas others were expressed at similar levels in both sexes. The gene encoding Anti-Müllerian hormone (AMH) was expressed in both sexes prior to and during sexual differentiation of the gonads, with levels of expression consistently higher in males than in females. SOX9 expression was male-specific, and was up-regulated after the detection of AMH transcripts. SOX3 expression was observed prior to clear SOX9 expression and was up-regulated in both sexes at the onset of gonadal sex differentiation (but declined later in development). The WT1 gene was highly expressed in both sexes, whereas SF1 expression was clearly higher in developing ovaries compared to testes. DAX1 transcripts were observed in both sexes at all stages examined, but expression appeared somewhat higher in developing ovaries. These expression profiles are analysed in terms of current theories of vertebrate sex determination.

Analysis of erythropoietin and erythropoietin receptor genes expression in cattle during acute infection with Trypanosoma congolense

Acute Trypanosoma congolense infection induced moderate, transient anemia in N’Dama cattle (trypanotolerant) and severe anemia in Boran cattle (trypanosusceptible). Erythropoietin receptor (EpoR) was cloned and sequenced from the two breeds of cattle. A single position mutation of Tyr in the Boran to His in the N’Dama predicted amino acid sequence was revealed. The mRNA transcription of erythropoietin (Epo) in kidneys and EpoR in the bone marrow of infected cattle was determined by competitive reverse transcription and the polymerase chain reaction (RT-PCR). Though Epo mRNA transcription increased in the kidneys during infection, the increase was not significantly different ( p > 0.05) between the two breeds of infected cattle. The level of EpoR transcripts in the bone marrow of infected N’Damas was significantly higher ( p < 0.05) than that detected in the marrows from infected Boran cattle. While infection seem to increase levels of transcription of IL-1α and β, and TNFα in kidneys from both Boran and N’Dama cattle, no significant difference was detected in the level of mRNAs of these cytokines in the kidney from the two breed of cattle. The amount of IFNγ mRNA transcripts were not changed with infection in N’Dama cattle, while on the contrary a significant higher levels of IFNγ was found in kidneys from infected Boran cattle as compared to the other groups. A significant ( p < 0.05) increase in the levels of IL-1α and β, and IFNγ mRNA transcripts were detected in the marrows of infected Borans as compared to the infected N’Dama cattle. In this study the increase in the level of TNFα mRNA in the marrows of the two infected breeds was not different. This implies there is no negative effect of TNFα on hematopoiesis during acute infection. These findings suggest that the levels of Epo and EpoR in the infected Boran cattle were inadequate for their degree of anemia, which might be due in part to high expression of IFNγ during acute infection with T. congolense.

Identification of mRNA transcripts and immunohistochemical localization of Na/H exchanger isoforms in gerbil inner ear

Recent physiological and pharmacological studies have implicated involvement of the Na/H exchanger (NHE) in regulating inner ear ion homeostasis, but the cellular distribution of this membrane transporter remains unknown. Here reverse transcription and the polymerase chain reaction (RT-PCR) were employed to screen adult gerbil inner ears for mRNA transcripts encoding the four best characterized isoforms of NHE. PCR products spanning selected segments of NHE mRNAs were cloned and sequenced. The putative housekeeping gene NHE-1 was found to be expressed and the 459 bp product shared 98.7% amino acid homology with rat sequence. NHE-2, NHE-3 and NHE-4 cDNA transcripts likewise were detected and the PCR products shared 100, 99.4 and 88.9% amino acid homology, respectively, with their rat counterparts. In addition, the cellular distribution of NHE isoforms 1 and 3 was mapped in the gerbil inner ear by immunostaining with polyclonal antisera against rat antigens. In the cochlea, the antiserum against NHE-1 reacted strongly at the basolateral membrane of strial marginal cells as well as with inner and outer hair cells and spiral ganglion neurons. Less intense staining for NHE-1 was present in subpopulations of fibrocytes in the spiral limbus and in inferior and superior areas of the spiral ligament. In the vestibular system dark and transitional cells expressed abundant NHE-1 as did hair cells and vestibular ganglia neurons. Immunostaining with the antiserum against NHE-3 was limited to the apical surface of marginal cells in the stria vascularis. Based on these data, NHE-1 likely functions primarily to maintain intracellular pH levels in cells where it is found in high abundance. NHE-3, on the other hand, possibly participates in the vectorial transcellular movement of Na + by strial marginal cells thus helping to maintain the extremely low Na + level in cochlear endolymph.

Expression ofCYP2E1during Embryogenesis and Fetogenesis in Human Cephalic Tissues: Implications for the Fetal Alcohol Syndrome

Reverse transcription and the polymerase chain reaction (RT-PCR) with oligonucleotide primers designed to target cDNA nucleotides 1241-1357 corresponding to exons 8 (3′ end) and 9 (5′ end) in human genomicCYP2E1detected consistently strong signals in 9 of 10 prenatal human brains. Cephalic tissues analyzed were between 54 and 78 days of gestation. RT-PCR signals for expression ofCYP2E1in corresponding human hepatic or adrenal tissues were weaker or, with only 2 exceptions, undetectable. Attempts to approximate levels of P4502E1 mRNA with Northern blots and RNase protection assays indicated that levels in human prenatal whole brain tissues tended to increase as a function of gestational age but, at the early stages investigated, were far lower than the constitutive levels in hepatic tissues of adult humans or male rats. Localized, P4502E1-dependent cephalic bioactivation of ethanol, with associated generation of several reactive chemical species, could contribute significantly to the etiology of neuroembryotoxic effects of prenatal ethanol exposure.

Immunohistochemical localization of the Na-K-Cl co-transporter (NKCC1) in the gerbil inner ear.

We mapped the cellular and subcellular distribution of the Na-K-Cl co-transporter (NKCC) in the adult gerbil inner ear by immunostaining with a monoclonal antibody (MAb T4) generated against human colon NKCC. Heavy immunolabeling was seen in the basolateral plasma membrane of marginal cells in the stria vascularis and dark cells in the vestibular system. Subpopulations of fibrocytes in the cochlear spiral ligament and limbus and underlying the vestibular neurosensory epithelium also stained with moderate to strong intensity, apparently along their entire plasmalemma. Because MAb T4 recognizes both the basolateral secretory (NKCC1) and the apical absorptive (NKCC2) isoforms of the co-transporter, we employed reverse transcription and the polymerase chain reaction (RT-PCR) to explore isoform diversity in inner ear tissues. Using NKCC1 and NKCC2 isoform-specific PCR primers based on mouse and human sequences, only transcripts for NKCC1 were detected in the gerbil inner ear. The presence of abundant NKCC1 in the basolateral plasmalemma of strial marginal and vestibular dark cells confirms conclusions drawn from pharmacological and physiological data. The co-expression of NKCC1 and Na,K-ATPase in highly specialized subpopulations of cochlear and vestibular fibrocytes provides further evidence for their role in recycling K+ leaked or effluxed through hair cells into perilymph back to endolymph, as postulated in current models of inner ear ion homeostasis.

Influence of hypoxia on vascular endothelial growth factor and chorionic gonadotrophin production in the trophoblast-derived cell lines: JEG, JAr and BeWo

Growth of trophoblast tissue in early pregnancy is rapid and accomplished in an unusually hypoxic environment. Hypoxia has been reported to upregulate mRNA production of vascular endothelial growth factor (VEGF), and VEGF receptors have been found on trophoblast cells. These observations suggest that VEGF may have an important role in early placentation. This study examines the influence of hypoxia on both the production of the VEGF message and protein and on the production of human chorionic gonadotrophin (hCG) protein by the cell lines JEG, JAr and BeWo. Cells were grown under normoxic and hypoxic conditions for 72 h. The average oxygen tension in the culture media of the hypoxic cultures (6-7 kPa) was significantly less than in the normoxic cultures (19-21 kPa). RNA was extracted and message for VEGF(121), VEGF(165) and VEGF(189) found in all cell lines by reverse transcription and the polymerase chain reaction (RT-PCR). These messages were upregulated by hypoxia; findings confirmed by competitive PCR for VEGF and expression of the house keeping gene GAPDH. hCG and VEGF were measured by immunoassay. Hypoxia resulted in an increase in VEGF production (P<0.05) but had inconsistent effects on hCG production. In some experiments the absolute concentrations of hCG and VEGF in the culture media were noted to be significantly correlated (r>0.5, P<0.05). In addition to its role in angiogenesis, VEGF may have direct effects on trophoblast cells encouraging proliferation and invasion. These effects may be regulated in part through oxygen supply and hCG.

Synthesis of β 2 glycoprotein 1 by the human placenta

Autoantibodies to negatively charged phospholipids (aPL) can cause fetal loss, including recurrent miscarriage and stillbirth. The immunopathogenic mechanism by which this occurs is unknown, but these antibodies are known to bind phospholipid via a protein cofactor, beta2 glycoprotein 1. This protein is localized on placental syncytiotrophoblast and enables the binding of aPL from maternal blood. In this study, reverse transcription and the polymerase chain reaction (RT-PCR) was used on mRNA isolated from normal human placental villous tissue and from human choriocarcinoma cell lines (Jeg-3, BeWo and JAr) to demonstrate that placental cells themselves synthesize beta2 glycoprotein 1 transcripts. Protein production was confirmed by immunoblotting experiments. Previous immunohistochemical studies were extended to demonstrate that beta2 GP1 is localized to extravillous cytotrophoblast in addition to syncytiotrophoblast. Production of beta2 glycoprotein 1 by fetal trophoblast indicates this protein is likely to have a physiological function in the placenta, and hence aPL may induce their pathological effect in pregnancy by inhibiting the function of placental beta2 glycoprotein 1.

Sequence of mouse 17beta-hydroxysteroid dehydrogenase type 3 cDNA and tissue distribution of the type 1 and type 3 isoform mRNAs.

The enzyme 17beta-hydroxysteroid dehydrogenase (17betaHSD) interconverts 17-ketosteroids and 17beta-hydroxysteroids. Five isoforms of the enzyme have been identified in the human, two of which (types 1 and 3) have been shown to catalyse the reduction reaction preferentially. The cDNA encoding mouse 17betaHSD type 3 was isolated from testis cDNA using the RACE technique and primers based on the human sequence. The mouse protein is 305 amino acids in length which is five short of the human protein with four of these amino acids missing at the N-terminus. The predicted amino acid sequence is 72.5% identical and 94.8% similar to the human sequence. Tissue distribution of mRNA encoding both types 1 and 3 17betaHSD was studied using reverse transcription and the polymerase chain reaction (RT-PCR). Highest levels of type 1 mRNA were found in the ovary whereas highest levels of type 3 were in the testis. All other tissues tested contained mRNA encoding both isoforms of the enzyme although levels were markedly lower than in the gonads.

Expression of follicle-stimulating hormone-receptor mRNA alternate transcripts in bovine granulosa cells during luteinization in vivo and in vitro

In cattle, as in many other species, formation of the corpus luteum is associated with loss of sensitivity to FSH. To determine whether this is associated with changes in FSH-receptor mRNA levels or alternate splicing of the primary transcript, reverse transcription and the polymerase chain reaction (RT-PCR) were used to examine transcripts during granulosa cell luteinization in vivo and in vitro. Using RT-PCR and Southern blotting, three alternate transcripts of the FSH-receptor were found consistently in bovine granulosa cells. The largest transcript encoded the full-length receptor while the other transcripts lacked either exon 9 or exons 4, 5 and 9. One day after luteinization in vivo, full-length FSH-receptor mRNA was detectable at low levels in the newly-formed corpus luteum. By day 3, however, no full-length transcripts were detectable in the corpus luteum. In contrast, when primers were used which amplify only the extracellular domain, FSH-receptor transcripts were detectable in all corpora lutea tested up to mid-cycle. In granulosa cells, luteinized in vitro, there was a similar loss of full-length FSH-receptor transcripts after day 1 but continued expression of transcripts encoding the extracellular domain. Results show that granulosa cell luteinization in cattle is associated with a change in splicing of the FSH-receptor primary transcript such that after luteinization only shortened transcripts coding for the extracellular domain are detectable. This process resembles, in reverse, changes in FSH-receptor transcript splicing during development of the gonads.

Rapid detection and identification of odontoglossum ringspot virus by polymerase chain reaction amplification

The odontoglossum ringspot Tobamovims (ORSV) movement and coat proteins genes were selected for the design of oligonucleotide primers for amplification of a 1,085 bp fragment. A combined assay of reverse transcription and the polymerase chain reaction (RT-PCR) was performed with 20-mer ORSV-specific primers and crude nucleic acid extracts from virus-infected orchids for rapid detection of the virus. The lowest concentration of template viral RNA required for detection was 10 fg. The RT-PCR is a 10 3 times more sensitive, reproducible and time-saving method than the enzyme-linked immunosorbent assay. No PCR product was observed when cymbidium mosaic potexvirus or a crude extract of healthy Cymbidium sp. were used as a template in RT-PCR with the same primers. The specificity of the primers was verified using other tobamoviruses RNAs.

Rapid detection and identification of odontoglossum ringspot virus by polymerase chain reaction amplification.

The odontoglossum ringspot Tobamovirus (ORSV) movement and coat proteins genes were selected for the design of oligonucleotide primers for amplification of a 1,085 bp fragment. A combined assay of reverse transcription and the polymerase chain reaction (RT-PCR) was performed with 20-mer ORSV-specific primers and crude nucleic acid extracts from virus-infected orchids for rapid detection of the virus. The lowest concentration of template viral RNA required for detection was 10 fg. The RT-PCR is a 10(3) times more sensitive, reproducible and time-saving method than the enzyme-linked immunosorbent assay. No PCR product was observed when cymbidium mosaic potexvirus or a crude extract of healthy Cymbidium sp. were used as a template in RT-PCR with the same primers. The specificity of the primers was verified using other tobamoviruses RNAs.