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At least 865 records · Page 48Linked to original sources
PubMed2001

Gene expression in a pure population of odontoblasts isolated by laser-capture microdissection.

Studies of odontoblast differentiation and function have been limited due to difficulties in obtaining sufficient numbers of intact cells. We describe a novel approach of laser-capture microdissection to obtain homogenous populations of pre-odontoblasts and odontoblasts from tissue sections of mouse molar cusp tips. Fixation, processing, and staining conditions were assessed for the optimal retrieval of total RNA from microdissected odontoblasts. Fluorometric assays and RT-PCR analysis of alpha1(I) collagen, dentin sialophosphoprotein (Dspp), and osteocalcin (OC) confirmed that the total RNA from three-day-old captured odontoblasts was sufficient in quantity and quality. Odontoblast-specific gene expression was studied by RT-PCR analysis performed in a single streptavidin-coated tube. At E15.5, Days 0 and 3, gene expression in laser-captured odontoblasts resembled that seen in vivo by in situ hybridization. The use of LCM is thus a valuable means of retrieving quality RNA from discrete populations of odontoblasts at different stages of dentinogenesis.

Animals↗
PubMed1992

Detection of prolactin receptor (PRL-R) mRNA in the rat hypothalamus and pituitary gland.

Prolactin receptor (PRL-R) mRNAs exist in several tissues where prolactin is known to act including the liver, testes, prostate, ovary, mammary gland, adrenal gland and kidney. PRL also acts at the level of the hypothalamus and pituitary gland to feed back and regulate its own secretion and the secretion of other anterior pituitary hormones. Therefore, we hypothesized that PRL-R mRNA would exist in these target tissues as well. Total RNA was extracted from rat anterior and medial basal hypothalamus, anterior and posterior pituitary gland, cerebral cortex, skeletal muscle and liver. After reverse transcribing total RNA with Murine-MLV reverse transcriptase and random or oligo(dT) pmers, the polymerase chain reaction (PCR) was performed. PCR products were then analyzed by ethidium bromide staining. Using primers that flanked the coding region for the extracellular binding domain we detected PRL-R mRNA in the anterior and medial basal hypothalamus, anterior and posterior pituitary gland, as well as in the liver, but not in the cerebral cortex or skeletal muscle. In addition, when we used primers that distinguish the long and short forms of the PRL-R mRNA, both forms of the PRL-R mRNA were detectable in the same tissues. Our data suggest that PRL may feed back at the level of the hypothalamus and pituitary gland through the same short and/or long PRL-R mRNA that mediate PRL action in the peripheral tissues.

Animals↗
PubMed1999

Modulation of myocilin/TIGR expression in human trabecular meshwork.

PURPOSE: To study factors that modulate myocilin/trabecular meshwork inducible glucocorticoid response protein (TIGR) mRNA expression in human trabecular meshwork (TM). METHODS: mRNA from fresh TM of four human donors, from perfused anterior segment organ cultured TM of three donors, and from four primary TM cell lines of different donors was isolated. The full length cDNA of myocilin/TIGR was cloned from TM mRNA using a polymerase chain reaction approach and used as probe for northern blot analysis hybridization. Trabecular meshwork cell cultures were treated with transforming growth factor (TGF)-beta1 (1 ng/ml), dexamethasone (10(-7) M), and mechanical stretch (10%). RESULTS: mRNA for myocilin/TIGR could be readily detected by northern blot analysis hybridization in 2 to 3 microg of total RNA from all fresh and all organ-cultured TM samples. In contrast, no mRNA for myocilin/TIGR could be detected in 20 microg of total RNA isolated from three different primary TM cell lines. Only one TM cell line had a baseline expression of myocilin/TIGR, which was 35- to 55-fold lower than that of fresh or organ-cultured TM samples. Treatment of TM cell cultures with dexamethasone for 1 day markedly increased expression of myocilin/TIGR mRNA, an effect that was even more pronounced after 3 days of treatment. Treatment with TGF-beta1 for 24 hours had no effect; however, after 3 and 12 days of treatment a 3.8- and 4-fold increase in myocilin/TIGR mRNA expression was observed. Expression of myocilin/TIGR mRNA was also increased after 10% mechanical stretch; however, in contrast to the effects of TGF-beta-1, this effect was observed much earlier (8-24 hours) after treatment. CONCLUSIONS: Dynamic mechanical stimuli maintain myocilin/TIGR expression in TM in situ and lack of these stimuli in monolayer cell cultures might be involved in downregulation of myocilin/TIGR expression.

Adult↗
PubMed1992

[The expression of oncogenes in the development of human brain].

Seven oncogenes (c-myc, L-myc, N-myc, v-erbB, c-fos, Ha-ras and mos) were used as the probe to detect the total RNA of every part (cerebellum, temporal folium, frontal folium and occipital folium) in human brain from 2 cases of 6 mon fetus and the total RNA in fetal development (4 mon, 5 mon, 6 mon, 7 mon, and newborn) of human brain tissue by RNA dot hybridization analysis. The results showed that most oncogenes were expressed at high level in cerebellum and occipital folium of 6 mon fetus and at low level in temporal folium; there was similar expression of c-myc, L-myc and N-myc in fetal development of human brain tissue, and the highest level was in the 5 mon fetal brain. There was no expression of erbB gene in normal human brain. The high level expression of Ha-ras and c-fos genes was in the middle and later stages of fetal development. These results suggested that c-myc, L-myc, N-myc, erbB, Ha-ras, c-fos and mos genes might play an important role in the fetal development of human brain.

Animals↗
PubMedDate not supplied

[Isolation and partial characterization of poly-(a)+mRNA of soybean grown in the presence of gibberellic acid].

Poly(A)+mRNA was prepared by chromatography on the oligo (dT)-cellulose column from total RNA of epicotyls of soybean grown on the medium with gibberellic acid. Poly(A)+mRNA was prepared by the same method from control plants. Total RNA, poly-A and poly(A)+mRNAs were analyzed by electrophoresis on 1.5% agarose gel under denaturing conditions. The poly(A)+mRNA distribution was shown to be of polydispersional character, cDNA was synthesized by reverse transcription. Basing on cDNA-poly(A)+mRNA hybridization a conclusion is drawn that gibberellic acid may activate new types of mRNAs and, probably, increases the synthesis of some poly(A)+mRNA populations.

Culture Media↗
PubMed1989

The time course of liver DNA and RNA alterations in acute experimental pancreatitis in rats--a possible mechanism of prostacyclin (PGI2) protection.

The mechanism of the liver damage in acute pancreatitis remains unsolved. The aim of this study was to evaluate RNA and DNA changes in the liver in Na-taurocholate pancreatitis in rats, with consideration being given to the protective effect of prostacyclin (20 micrograms.kg.24 h intraperitoneally). Total RNA increased both in treated and untreated rats after 24 hrs and even more after 48 hrs. After 48 hrs this increase was 35% higher in treated than in untreated rats. The RNA/DNA ratio paralleled the increase of total RNA. In 24 hrs incorporation of 3H-uridine into RNA increased 145% in untreated, and in 48 hrs 50% in treated rats. After 7 days 60% of treated animals survived as compared with 20% in the untreated group. The evident changes in RNA content, RNA/DNA ratio and RNA synthesis suggest a role for nucleic acid disturbances in the liver injury in acute pancreatitis. The beneficial effect of prostacyclin might, in part, be dependent on its effect on nucleic acid metabolism.

Acute Disease↗
PubMed1991

A comparison of substance P peptide and preprotachykinin mRNA levels during development of rat medullary raphe and neostriatum.

Substance P (SP) and the mRNA coding for its precursor peptide, preprotachykinin (PPT), were measured in medullary raphe nuclei (MRN) and neostriatum (NS) over development in order to determine (1) whether PPT mRNA levels correlate with peptide development, and (2) whether changes in PPT mRNA might help to account for the apparent decline in SP seen immunohistochemically in certain brain areas postnatally. Total RNA was quantified in dissected tissue pieces using the sensitive orcinol reaction. When MRN PPT mRNA levels measured by Northern blot analysis were adjusted to total RNA levels, PPT mRNA per MRN increased over development with a profile similar to that seen for SP peptide. Moreover, both peptide and mRNA levels exhibited a similar decline after postnatal day 15. Therefore, developmental regulation of SP biosynthesis in the MRN may, in part, explain previous evidence documenting a postnatal decline in SP there. In the NS, SP peptide and PPT mRNA increased with a similar profile from E18 through the first postnatal week. Thereafter, SP increased less rapidly than its mRNA, indicating incongruities in prohormone message and processed peptide in the NS.

Animals↗
PubMed2004

Development and application of a real-time quantitative PCR assay for determining CYP1A transcripts in three genera of salmonids.

The expression of CYP1A (cytochrome P4501A) can be induced by a large array of aromatic and organic compounds in teleost fishes. We developed a real-time quantitative PCR assay useful for measuring beta-naphthoflavone (BNF) induction of liver CYP1A mRNA in four salmonid species. First, to obtain necessary information for the design of a cRNA standard, full-length CYP1A cDNA sequences were determined for two Salvelinus species, lake trout (S. namaycush) and brook trout (S. fontinalis). Each cDNA was found to share the same characteristics with known CYP1A sequences of Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss): a start codon, conserved heme-binding region, putative poly-adenylation signal, stop codon, relatively long 3'-untranslated region (UTR; >1 kb), and a protein length of 523 amino acid residues. The brook trout and lake trout CYP1A cDNA's were 2636 and 2672 base pairs (bp) in length and shared greater than 97% coding region sequence identity with Atlantic salmon and rainbow trout CYP1A's. Next, using the generated sequence information, we developed a CYP1A-specific real-time quantitative PCR assay. Primers and a fluorescent-labeled probe were designed from a 68 bp region that was found to be conserved among salmonid CYP1A genes. The assay was designed to allow for simultaneous comparison of CYP1A expression among each experimental group. Finally, groups (n = 4-8) of hatchery-raised Atlantic salmon, brook trout, lake trout, and rainbow trout were given an intraperitoneal injection of a corn oil control, 25 mg kg(-1) BNF, or 50 mg kg(-1) BNF and sacrificed after 48 h. Liver tissue was collected and CYP1A mRNA levels were estimated. In all species, BNF treated fish showed 1.8-3.0 orders of magnitude higher CYP1A than control fish. The CYP1A induction levels were not different in fish treated with both dosages. Mean base levels of CYP1A expression ranged from 7.24 x 10(6) (rainbow trout) to 1.05 x 10(7) (brook trout) transcripts microg(-1) total RNA. Mean induced levels of CYP1A expression ranged from 1.07 x 10(8) (lake trout) to 1.05 x 10(9) (brook trout) trancripts microg(-1) total RNA.

Amino Acid Sequence↗
PubMed2002

Gene expression of pro-inflammatory cytokines and chemokines in mouse eye infected with Pseudomonas aeruginosa.

Ocular infection with Pseudomonas aeruginosa triggers extensive host inflammatory response and corneal damage. The purpose of present study was to investigate the gene expression of pro-inflammatory mediators interleukin (IL)-1 beta, IL-6, tumour necrosis factor-alpha (TNF-alpha),macrophage inflammatory protein (MIP)-2 and cytokine-induced neutrophil chemoattractant (KC) in the mouse eye challenged with P. aeruginosa. Scratched mouse corneas were infected with three phenotypes of P. aeruginosa individually. Total RNA was extracted from mouse eyes at 4 h, 8 h,16 h and 24 h post-challenge. Single stranded cDNA was synthesized from total RNA by reverse transcription and then subjected to polymerase chain reaction (PCR) using specific primers for IL-1 beta, IL-6, TNF-alpha, MIP-2 and KC. Results revealed three patterns of cytokines and chemokines expression in response to ocular infection with three phenotypes of P. aeruginosa. Ocular infection with the invasive strain induced the highest levels of IL-1 beta, IL-6, MIP-2 and KC mRNA, followed by the infection with the cytotoxic strain. Ocular infection with the CLARE strain induced the lowest levels of IL-1 beta, IL-6, MIP-2 and KC mRNA. The expression of TNF-alpha mRNA was very low and irregular following P. aeruginosa challenge. These data indicate that over-expression of pro-inflammatory cytokines and chemokines may represent a vigorous immune response and therefore may contribute to corneal damage during P. aeruginosa infection.

Animals↗
PubMed1999

Androgen receptor mRNA expression in the rhesus monkey ovary.

Immunocytochemical detection of androgen receptors (ARs) in several compartments of the macaque ovary, including the germinal epithelium, follicle, and corpus luteum, suggests a role for androgens in modulating ovarian function via the classical receptor-mediated pathway. To examine AR mRNA expression in the rhesus monkey ovary, total RNA was isolated from whole ovaries, the germinal epithelium-enriched cortical and medullary compartments of the ovary, and corpora lutea from early (d 3-5), mid (d 6-8), mid-late (d 10-12), and late (d 13-15) stages of the luteal phase of the menstrual cycle. RNA was also obtained from luteinized granulosa cells from monkeys receiving gonadotropin treatment to stimulate the development of multiple ovarian follicles. After reverse transcription of total RNA using oligo-dT as a primer, polymerase chain reaction (PCR) was used to amplify a unique 329 bp segment of the monkey AR hormone-binding region. Reverse transcriptase (RT)-PCR products of the expected size were detected in all ovarian and control tissues. Sequence analysis of the AR cDNA from the macaque ovary revealed 99% nucleotide homology and 100% predicted amino acid homology to the cDNA for the hormone-binding region of human AR. Northern analysis demonstrated the presence of a major AR mRNA species at 9.5 kb in corpus luteum, luteinized granulosa cells, and prostate, with additional bands detected in the corpus luteum and prostate at 7.9 and 3.4 kb, respectively. A sensitive RNase protection assay was used to examine AR mRNA levels in ovarian tissues and showed AR mRNA expression throughout the life-span of the corpus luteum. Thus, detection of AR mRNA in the primate ovary, including the periovulatory follicle and corpus luteum, supports the concept that these tissues are targets for receptor-mediated androgen action during the menstrual cycle.

Animals↗
PubMed2002

RNA amplification results in reproducible microarray data with slight ratio bias.

Microarray expression analysis demands large amounts of RNA that are often not available. RNA amplification techniques have been developed to overcome this prcblem, but limited data are available regarding the reproducibility and maintenance of original transcript ratios. We optimized and validated two amplification techniques: a modified in vitro transcription for the linear amplification of 3 microg total RNA and a SMART PCR-based technique for the exponential amplification of 50 ng total RNA. To determine bias between transcript ratios, we compared the expression profiles in mouse testis versus spleen between the two amplification methods and a standard labeling protocol, using microarrays containing 4596 cDNAs spotted in duplicate. With each method, replicate hybridizations were highly reproducible. However, when comparing the amplification methods to standard labeling, correlation coefficients were lower. Twelve genes that exhibited inconsistent or contradictory expression ratios among the three methods were verified by quantitative RT-PCR. The amplification methods showed slightly more discrepancies in the expression ratios when compared to quantitative RT-PCR results but were more sensitive in terms of detecting expressed genes. In conclusion, although amplification methods introduce slight changes in the transcript ratios compared to standard labeling, they are highly reproducible. For small sample size, in vitro transcription is the preferred method, but one should never combine different labeling strategies within a single study.

Blotting, Southern↗
PubMed1989

Elevation of a specific mRNA in longissimus muscle of steers fed ractopamine.

To address the hypothesis that some classes of growth promoters stimulate muscle protein synthesis in growing cattle, 23 crossbred steers were fed diets containing the phenethanolamine growth promoter ractopamine in a 140-d feeding trial. Steers received either no ractopamine, .18 or .36 mg.kg BW-1.d-1 ractopamine for 140 d or .36 or .72 mg.kg BW-1.d-1 ractopamine for 56 d. Longissimus muscle was obtained at slaughter and frozen in liquid N2. RNA was extracted by homogenization of pulverized frozen muscle in guanidinium isothiocyanate and centrifugation through cesium chloride. Polyadenylated mRNA was extracted by capture on oligo-dT columns. Ractopamine had no effect on total RNA or mRNA concentrations (P greater than .25). Hybridization of the RNA to a putative myosin light chain-1/3 (MLC-1/3) cDNA clone in a Northern blot indicated one heavy band (approximately 1 kb) with no evidence of extensive destruction of the RNA. A second, minor band (approximately 3 kb) also was observed in some samples. The MLC-1/3 cDNA clone was hybridized to 1- or 5-micrograms samples of total RNA, and the intensity of the resultant autoradiographs was quantified by laser densitometry. There was a statistical correlation between MLC-1/3 mRNA-micrograms RNA-1 and longissimus cross-sectional area (P less than .05) and average daily gain (P less than .025). The results suggest that ractopamine either increased the transcription of the putative MLC-1/3 gene and(or) increased the stability of MLC-1/3 mRNA in bovine longissimus muscle, either of which could result in an increase in specific myofibrillar protein synthesis.

Analysis of Variance↗
PubMed2004

Oligonucleotide microarray analysis of aminoallyl-labeled cDNA targets from linear RNA amplification.

Single-stranded long oligonucleotide-based (50- to 70-mer) microarrays offer several advantages over conventional cDNA microarrays. These include the easy preparation of the probes, low cost of array production, and low cross-contamination during probe handling. However, the application of oligonucleotide microarrays for the analysis of global gene expression with small amounts of total RNA using the conventional oligo(dT)-T7 promoter-based amplification is hampered by the single-stranded nature (sense strand) of oligonucleotide probes in microarrays. In this report, we describe modified RNA amplification methods generating antisense-labeled cDNA targets and a successful application for oligonucleotide microarray gene expression analysis. In the first round, mRNA was amplified linearly with oligo(dT)24T7-primed reverse transcription and in vitro transcription by T7 RNA polymerase. In the second round, random 9-mer T3 primers and T3 RNA polymerase were used to generate sense-strand amplified RNA (aRNA). Fluorescently labeled cDNA targets were generated from the aRNA and hybridized to the oligonucleotide microarrays. Our data show that the amplification provides highly reproducible results, as evidenced by a significant correlation between the amplified and nonamplified samples. We also demonstrate that amplification of RNA derived from laser-microdissected tumor samples reproduced the gene expression profiles that were obtained from total RNA isolated from the same samples.

Animals↗
PubMed1998

Fetal expression of GnRH and GnRH receptor genes in rat testis and ovary.

The identification of gonadal gonadotropin-releasing hormone receptors (GnRH-R) and evidence of direct inhibitory effects of GnRH agonists upon steroidogenesis in adult rat gonads, lend credence to a putative intragonadal role of a locally secreted GnRH or GnRH-like peptide. Using reverse transcription-polymerase chain reaction followed by Southern blot hybridization and sequencing, we identified, both in the ovary and in the testis of fetal and adult rats, a fully processed GnRH messenger RNA (mRNA), the sequence of which, in adult testis, was identical to that found in the hypothalamus. We also detected in the testis, but not in the ovary, a transcript containing the first intron. The ontogeny of GnRH and GnRH-R gene expression was studied in rat gonads from 14.5 to 21.5 days post-coitum (dpc), using dot blot hybridization of total RNA. During this period, the levels of cyclophilin mRNA normalized to total RNA remained unchanged. Thus, we used cyclophilin as an internal standard. GnRH mRNA was detected in the ovary at 18.5 dpc, four days later than in the testis, and similar levels were found in both sexes at birth. GnRH-R mRNA was present at 14.5 dpc in the testis and at 15.5 dpc in the ovary, with the levels at 21.5 dpc being 2.4 times higher in the testis than in the ovary. GnRH and GnRH-R mRNA levels increased in both sexes in late fetal development, but this increase appeared two days sooner in the ovary compared with the testis, thus supporting the hypothesis that expression of the GnRH and GnRH-R genes is regulated in a sex-dependent manner during fetal development. In all cases, expression of GnRH and GnRH-R preceded gonadotropin receptors in the gonads and initiation of gonadotropin secretion by the pituitary.

Animals↗
PubMed2006

Systematic comparison of the T7-IVT and SMART-based RNA preamplification techniques for DNA microarray experiments.

BACKGROUND: Small biological samples obtained from biopsies or laser microdissection often do not yield sufficient RNA for successful microarray hybridization; therefore, RNA amplification is performed before microarray experiments. We compared 2 commonly used techniques for RNA amplification. METHODS: We compared 2 commercially available methods, Arcturus RiboAmp for in vitro transcription (IVT) and Clontech BD SMART for PCR, to preamplify 50 ng of total RNA isolated from mouse livers and kidneys. Amplification factors of 3 sequences were determined by real-time PCR. Differential expression profiles were compared within and between techniques as well as with unamplified samples with 10K 50mer oligomer-spotted microarrays (MWG Biotech). The microarray results were validated on the transcript and protein levels by comparison with public expression databases. RESULTS: Amplification factors for specific sequences were lower after 2 rounds of IVT than after 12 cycles of SMART. Furthermore, IVT showed a clear decrease in amplification with increasing distance of the amplified sequences from the polyA tail, indicating generation of smaller products. In the microarray experiments, reproducibility of the duplicates was highest after SMART. In addition, SMART-processed samples showed higher correlation when compared with unamplified samples as well as with expression databases. CONCLUSIONS: Whenever 1 round of T7-IVT does not yield sufficient product for microarray hybridization, which is usually the case when <200 ng of total RNA is used as starting material, we suggest the use of SMART PCR for preamplification.

3' Untranslated Regions↗
PubMed1995

The 5-lipoxygenase pathway in cultured human intestinal epithelial cells.

Leukotrienes (LTs), the 5-lipoxygenase (5-LOX) metabolites of arachidonic acid, have roles in many biological processes relevant to the gastrointestinal tract, including intestinal inflammation. We screened two well-known human intestinal epithelial cell lines, HT29 and Caco-2, for evidence of LT-associated enzyme transcripts and LT synthesis. Northern blot analysis of total RNA from both intestinal lines demonstrated high levels of transcripts for LTA4 hydrolase, a multisubstrate enzyme that converts the 5-LOX metabolite, LTA4, to LTB4. With total RNA, the 5-LOX transcript was detected only in HT29. Caco-2 failed to show 5-LOX message even with poly A-containing RNA, although the transcript could be amplified with the polymerase chain reaction. Messenger RNA for FLAP, the 5-lipoxygenase-activating protein, was detectable in both cell lines, but only with poly A-containing RNA. In a sonicated cell preparation, HT29, but not Caco-2, revealed detectable levels of 5-HETE and LTB4. These results suggest that certain intestinal epithelial cells possess a limited capacity to synthesize LTs.

5-Lipoxygenase-Activating Proteins↗
PubMed2003

Quantitation of changes in gene expression of norepinephrine biosynthetic enzymes in rat stellate ganglia induced by stress.

Enzymes involved in catecholamine synthesis are present in the highest concentration in the adrenal medulla, however they were found also in other, mainly nervous tissues. The aim of our study was to quantify the exact concentration of tyrosine hydroxylase (TH) and dopamine-ss-hydroxylase (DBH) mRNA in rat stellate ganglia under control conditions and at different intervals after exposure to immobilization stress (IMO). In rats immobilized once for 2h, we determined TH and DBH mRNA in different time intervals up to 22 h after the end of the stress stimulus. TH immunoreactive protein levels were also determined in stellate ganglia. TH and DBH mRNA levels were quantified by RT-competitive-PCR. In stellate ganglia, the concentration of TH mRNA was 17+/-1.6 amol/microg of total RNA, which is approximately 30-times lower than in the adrenal medulla. The concentration of DBH mRNA in the stellate ganglia was 2601+/-203 amol/microg of total RNA, which is the concentration similar to adrenal medulla, but is 150-times higher than concentration of TH mRNA in stellate ganglia. After a single 2-h immobilization the highest elevation of TH and DBH mRNA levels was measured 22 h after the termination of the stress stimulus. Repeated immobilization (7 days, 2h daily) did not produce further increase in TH and DBH mRNA levels compared to already elevated levels in adapted control group (immobilized for 6 days, 2h daily and decapitated 22 h later). Levels of TH protein were significantly changed only after the repeated immobilization. This study compared for the first time the precise amounts of TH and DBH mRNA in rat stellate ganglia under control conditions and after immobilization stress, and indicates large differences in their concentration. TH and DBH mRNA concentrations in stellate ganglia are markedly elevated for a prolonged period of time after termination of the stress stimuli.

Animals↗
PubMed1998

Expression of type XVII collagen alpha 1 chain mRNA in the mouse heart.

The type XVII collagen alpha 1 chain has been identified as a component of the type I hemidesmosome, and is thus thought to play a role in extracellular matrix (ECM) maintenance and signal transduction between the cell and the ECM. We examined the expression of type XVII collagen alpha 1 chain mRNA in the mouse heart by Northern blot analysis and determined the sequential changes of its expression in different developmental stages of the heart using the reverse transcriptase-polymerase chain reaction (RT-PCR) method. Northern blotting: Total RNA was extracted from 10 adult mouse hearts by the guanidine/cesium method. Hybridization was performed with mouse cDNA for alpha 1 (XVII) collagen. RT-PCR: Total RNA was extracted from 7 embryos, 4 neonates and 8 adult mice. Reverse transcription was performed using oligo-dT primer and MMLV. Amplification was carried out in alpha 1 (XVII) collagen and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). GAPDH served as an internal control. Northern blotting revealed a 5.6 kb signal that was identical to that of the alpha 1 (XVII) of skin and transformed keratinocyte reported previously. The sequences of the PCR products were also identical to those reported. The normalized expression ratios of alpha 1 (XVII) were 0.91 +/- 0.20 in the embryonic heart, 0.36 +/- 0.20 in the neonatal heart and 0.96 +/- 0.21 in the adult heart. In conclusion, we identified the expression of type XVII collagen alpha 1 chain mRNA in the mouse heart, suggesting that the type I hemidesmosome is located in the heart. The results of the RT-PCR at different developmental stages of the heart suggest that type XVII collagen contributes not only to cardiogenesis in the embryonic stage but also to maintenance of architecture and function in the adult heart.

Angiotensin-Converting Enzyme Inhibitors↗