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At least 289 records · Page 16Linked to original sources
PubMed2001

The expression of thyrotrophin-releasing hormone receptor 1 messenger ribonucleic acid in human pituitary adenomas.

OBJECTIVE: Thyrotrophin-releasing hormone (TRH) paradoxically induces the release of growth hormone (GH) when injected intravenously into acromegalic patients, although the mechanism of this action is unknown at present. Several research groups have reported that the level of TRH receptor-1 (TRHR-1) mRNA expression is variable in pituitary adenomas, and does not correlate with the degree of paradoxical GH response to TRH administration in a limited number of acromegalic patients. We aimed to compare the expression levels of TRHR-1 mRNA among various types of pituitary adenoma and to clarify whether these levels correlate with the degree of pituitary hormone response to TRH. PATIENTS: Pituitary adenoma tissue was obtained by surgery from 14 patients with acromegaly, four with prolactinomas, nine with nonfunctioning adenomas and one with a TSH-producing adenoma. METHODS: The level of human TRHR-1 mRNA expression in each adenoma was quantified using the competitive reverse transcription polymerase chain reaction (RT-PCR) METHOD: For amplification of a TRHR-1 cDNA fragment, a sense primer was designed according to the sequence in exon 2 and an antisense primer designed according to the sequence located at the region in exon 3 that does not encode for the alternative splicing-generated short form of TRHR-1 mRNA. RESULTS: TRHR-1 mRNA was detected in all pituitary adenomas examined and did not correlate with their size. The mean level of TRHR-1 mRNA expression was significantly lower in GH-producing adenomas than in prolactinomas and nonfunctioning adenomas (1.4 +/- 0.4 x 10(-2) attomol/microg total RNA, 10.7 3.4 x 10(-2) attomol/microg total RNA, and 7.2 +/- 3.3 x 10(-2) attomol/g total RNA, respectively). The ratio of plasma peak GH induced by TRH administration to the basal level of plasma GH in the patients with acromegaly correlated positively with the level of TRHR-1 mRNA expression in their GH-producing adenomas (r = 0.620, P = 0.0179). The responsiveness of plasma PRL and gonadotrophin to TRH in the patients with prolactinoma and nonfunctioning pituitary adenoma did not significantly correlate with the levels of TRHR-1 mRNA expression in their pituitary adenomas, respectively. CONCLUSIONS: The findings of the present study suggest that the level of TRHR-1 mRNA expression varies among different types of pituitary adenoma. Furthermore, in acromegaly, the responsiveness of plasma GH to TRH administration appears to at least partially depend on the level of TRHR-1 mRNA expression in the GH-producing pituitary adenoma.

Acromegaly↗
PubMed1997

PPAR-gamma gene expression is elevated in skeletal muscle of obese and type II diabetic subjects.

The peroxisome proliferator activated receptor PPAR-gamma has been identified as a nuclear receptor for thiazolidenediones, which are compounds with insulin-sensitizing properties in several tissues, including skeletal muscle. To determine whether this receptor is expressed and possibly involved in insulin action/resistance in skeletal muscle, PPAR-gamma mRNA abundance and its regulation by insulin were quantified in muscle tissue and cultures from lean and obese nondiabetic and type II diabetic subjects using competitive reverse transcription-polymerase chain reaction (RT-PCR). In muscle biopsy specimens, PPAR-gamma mRNA was elevated in obese nondiabetic and type II diabetic subjects (23.4 +/- 4.2 and 28.0 +/- 5.69 x 10(3) copies/microg total RNA, respectively; both P < 0.05) compared with lean nondiabetic control subjects (9.4 +/- 2.3 x 10(3) copies/microg total RNA). Significant positive correlations were present among skeletal muscle PPAR-gamma mRNA levels, BMI (r = 0.67, P < 0.01), and fasting insulin concentration (r = 0.76, P < 0.001). PPAR-gamma mRNA levels were also elevated in muscle cultures from type II diabetic subjects compared with lean nondiabetic control subjects (330.1 +/- 52.9 vs. 192.1 +/- 27.0 x 10(3) copies/microg total RNA, P < 0.05). Insulin stimulation of muscle tissue (by hyperinsulinemic-euglycemic clamp for 3-4 h) or muscle cultures (30 nmol/l for 120 min) stimulated PPAR-gamma mRNA expression up to fourfold (10.0 +/- 2.7 to 41.3 +/- 7.4 x 10(3) copies/microg total RNA, P < 0.05, and 174.9 +/- 56.9 to 268.2 +/- 78.6 x 10(3) copies/microg total RNA, P < 0.05, respectively). In summary, PPAR-gamma mRNA expression in human skeletal muscle is acutely regulated by insulin and is increased in both obese nondiabetic and type II diabetic subjects in direct relation to BMI and fasting insulinemia. We conclude that abnormalities of PPAR-gamma may be involved in skeletal muscle insulin resistance of obesity and type II diabetes.

Adult↗
PubMed2004

Evaluation of a linear amplification method for small samples used on high-density oligonucleotide microarray analysis.

High-density oligonucleotide microarray analysis has proven to be an excellent approach for gene expression profiling in human cancers. This technique assesses the expression of thousands of genes simultaneously, from at least 5 microg of total RNA per sample per experiment. This total RNA requirement poses a challenge when studying small, unique clinical samples, like biopsies. Recently, a new standardized protocol for small samples was released by Affymetrix, which includes a linear amplification step. To evaluate the impact of such amplification in the gene expression profiling of human ovarian cancer, we compared results obtained from 5 microg and 100 ng of total RNA from the same tumor sample, using the standard Affymetrix protocol and the new linear RNA amplification protocol, respectively. We identified a small bias in gene expression data caused by linear amplification, potentially due to shorter elongation products leading to misclassification of probe sets directed to the middle-5' region of the transcripts. Interestingly, the magnitude of the bias varied when different normalization and expression summary algorithms were used. However, this bias does not affect tumor gene expression profiling. Consequently, linear amplification may be of utility in cases of extremely low RNA recovery from critical and unique samples, such as small biopsies.

Cluster Analysis↗
PubMed2005

A robust method for the amplification of RNA in the sense orientation.

BACKGROUND: Small quantities of RNA (1-4 microg total RNA) available from biological samples frequently require a single round of amplification prior to analysis, but current amplification strategies have limitations that may restrict their usefulness in downstream genomic applications. The Eberwine amplification method has been extensively validated but is limited by its ability to produce only antisense RNA. Alternatives lack extensive validation and are often confounded by problems with bias or yield attributable to their greater biological and technical complexity. RESULTS: To overcome these limitations, we have developed a straightforward and robust protocol for amplification of RNA in the sense orientation. This protocol is based upon Eberwine's method but incorporates elements of more recent amplification techniques while avoiding their complexities. Our technique yields greater than 100-fold amplification, generates long transcript, and produces mRNA that is well suited for use with microarray applications. Microarrays performed with RNA amplified using this protocol demonstrate minimal amplification bias and high reproducibility. CONCLUSION: The protocol we describe here is readily adaptable for the production of sense or antisense, labeled or unlabeled RNA from intact or partially-degraded prokaryotic or eukaryotic total RNA. The method outperforms several commercial RNA amplification kits and can be used in conjunction with a variety of microarray platforms, such as cDNA arrays, oligonucleotide arrays, and Affymetrix GeneChip arrays.

Algorithms↗
PubMed1990

Mechanisms of total lymphoid irradiation-induced immunosuppression. II. Failure of con A-stimulated splenocytes from TLI-treated mice to express IL-2 and IL-2 receptor RNA.

Total lymphoid irradiation is a radiotherapy procedure used as an alternative immunosuppressive regimen in organ transplantation. Following TLI mature lymphocytes are depleted, and splenocytes do not proliferate to mitogens, produce IL-2, or express IL-2 receptors. We now show that mitogen stimulated splenocytes from TLI-treated mice do not secrete IL-2 protein by an IL-2 ELISA assay. Northern blot analysis and RNase protection assays reveal that TLI splenocytes do not make IL-2 RNA or IL-2 receptor RNA following mitogen stimulation. TLI splenocytes produce at least 1000 times less IL-2 RNA after Con A stimulation than normal splenocytes. TLI therapy resembles anti-CD4 therapy and CsA in that each results in an IL-2-"depleted" state.

Animals↗
PubMed2001

Angiotensin regulates endothelin-B receptor in rat inner medullary collecting duct.

Our recent studies showed that endothelin (ET)(B) receptors are downregulated in congestive heart failure. These changes in ET(B) receptor density can be prevented by angiotensin-converting enzyme inhibitors, suggesting a possible role for angiotensin. Using isolated inner medullary collecting ducts (IMCD), we examined the possibility that angiotensin-induced downregulation of ET(B) receptors is accompanied by a decrease in ET(B) receptor mRNA. Binding studies showed that overnight incubation with angiotensin II induced a downregulatiion of ET(A) and ET(B) receptors' density in IMCD by 39% and 29%, respectively. This downregulation in ET receptor density was abolished when IMCD was coincubated with angiotensin II and its receptor antagonist saralasin. Furthermore, when the cells were exposed to phorbol myristate acetate (PMA), it resulted in a reduction in ET(A) and ET(B) receptor binding sites by 41% and 34%, respectively, suggesting the involvement of protein kinase C (PKC). In isolated IMCD, ET-1 induced an increase in cyclic guanosine monophosphate (cGMP) accumulation (705 + 63 to 1,015 + 88 fmol/microg protein/5min, P <.01), and the ET-1-induced accumulation was attenuated in the presence of angiotensin II (641 + 45 to 809 + 46 fmol/microg protein/5min, P <.01). Using competitive polymerase chain reaction (PCR) method, we also observed downregulation of ET(A) and ET(B) receptors mRNA in IMCD treated with angiotensin II (ET(A), 1.09+0.11 v 0.77 + 0.07 amol/microg of total RNA, P <.01; ET(B), 14.80 + 1.95 v 8.65 + 0.67 amol/microg of total RNA, P <.01). The addition of a PKC inhibitor abolished the downregulation of ET(A) and ET(B) receptor mRNA induced by angiotensin II (ET(A), 1.25 + 0.07 v 1.19 + 0.06 amol/microg of total RNA, not significant [NS]; ET(B), 14.36 + 0.83 to 13.68 + 0.64 amol/microg of total RNA, NS). These results suggest that angiotensin II-induced downregulation of ET(A) and ET(B) receptors mRNA is mediated by a mechanism involving PKC.

Angiotensin II↗
PubMed1983

Etiology of growth hormone deficiency in little, Ames, and Snell dwarf mice.

There are three recessive genes (little, Snell, and Ames) which cause dwarfism and GH deficiency in mice. These genes are nonallelic, and in the case of little mice, GH deficiency is isolated, while Snell and Ames dwarfs have deficiencies of GH as well as other anterior pituitary hormones. Previous reports in which restriction endonuclease analyses were used suggested that the GH genes are grossly intact in each of these types of dwarfs. In this report, ultrastructural studies of the anterior pituitary glands of little mice showed a deficiency or absence of secretory granules within somatotropes, while Snell and Ames dwarf pituitaries lacked identifiable somatotropes. Furthermore, there were deficiencies of GH precursor RNA and mRNA in total RNA from little pituitaries, while GH transcripts appeared to be absent in total RNA from Snell or Ames dwarf pituitaries. Thus, the primary defect in little mice may be in the production of GH transcripts, while GH deficiency in Snell and Ames dwarfs is probably due to defects other than alterations of the GH genes.

Animals↗
PubMed2005

Expression of endogenous and exogenous growth hormone (GH) messenger (m) RNA in a GH-transgenic tilapia (Oreochromis niloticus).

We have previously produced transgenic fish from crosses between a wild-type female tilapia (Oreochromis niloticus) and a G transgenic male. This line of growth-enhanced tilapia carries a single copy of a chinook salmon (s) growth hormone (GH) gene spliced to an ocean pout antifreeze promoter (OPA-FPcsGH) co-ligated to a carp beta-actin/lacZ reporter gene construct, integrated into the tilapia genome. Because little is known about the expression sites of transgenes, we have characterised the gene expression patterns of sGH and tilapia (t)GH in transgenic tilapia using a newly established real-time PCR to measure the absolute mRNA amounts of both hormones. The sGH gene, which was expected to be expressed mainly in liver, was also found to be expressed in other organs, such as gills, heart, brain, skeletal muscle, kidney, spleen, intestine and testes. However, in pituitary no sGH mRNA but only tGH mRNA was found. Tilapia GH mRNA in wild-type pituitary amounted to 226 +/- 30 pg/microg total RNA but in transgenics only to 187 +/- 43 pg/microg total RNA. Liver exhibited the highest level of sGH mRNA (8.3 +/- 2.5 pg/microg total RNA) but the extrahepatic sites expressed considerable amounts of sGH mRNA ranging from 4.1 +/- 2.0 pg/microg total RNA in gills to 0.2 +/- 0.08 pg/microg total RNA in kidney. The widespread expression of the sGH gene is assumed to be due to the tissue specificity of the type III AFP gene promoter. It is assumed that our transgenic experiments, which in contrast to some other approaches caused no obvious organ abnormalities, mimick the GH expression during ontogeny. Because sGH mRNA is expressed both in liver and in extrahepatic sites it may not only promote secretion and release of liver-derived (endocrine) IGF-I leading to an overall growth enhancement but also stimulate IGF-I expression within the different organs in a paracrine/autocrine manner and, thus, further promote organ growth.

Animals↗
PubMed1987

Simultaneous isolation of preparative amounts of RNA and DNA from Trichinella spiralis by cesium trifluoroacetate isopycnic centrifugation.

A rapid method for simultaneously banding preparative amounts of RNA and DNA from Trichinella spiralis muscle larvae by isopycnic centrifugation in cesium trifluoroacetate (CsTFA) is described. Larvae were homogenized in guanidinium isothiocyanate and the DNA, RNA, glycogen, and denatured protein components were isopycnically separated without prior purification. This procedure resulted in the isolation of nucleic acids suitable for molecular biological application. Agarose gel electrophoresis of gradient fractions indicated the separation of undegraded RNA and DNA where total RNA was of sufficient purity to efficiently direct in vitro translation of parasite protein and total DNA was greater than 20 kb in size and sensitive to restriction endonuclease digestion. Oligo (dT)-purified poly(A)+ mRNA was 3.6% of total RNA with greater than 18% conversion to cDNA.

Animals↗
PubMed1997

Synthesis of full-length potyvirus cDNA copies suitable for the analysis of genome polymorphism.

New methods facilitating the synthesis and amplification of full-length cDNA copies of single-stranded viral RNA genomes have been developed. A method is described for the efficient purification of potyviral RNA and total RNA from infected plants and it is shown that they can serve as templates for the efficient synthesis of a full-length, 10 kb long, genomic cDNA. Two different reverse transcriptases were used (AMV-RT and MMLV-RT); only the first reverse transcriptase produced a good quality, full-length cDNA using viral RNA as a template. Surprisingly, MMLV-RT allowed for the full-length cDNA synthesis on virions rather than viral RNA. The PVY cDNA, synthesized using either RNA or virions, can be amplified successfully by PCR with high yields of full-length products. Such products are good substrates for the study by RFLP of the total genome polymorphism of virus isolates.

DNA, Complementary↗
PubMed1982

Temperature dependence of RNA synthesis parameters in Escherichia coli.

For Escherichia coli B/r growing in glucose minimal medium, the following parameters of RNA synthesis remained invariant between 20 and 40 degrees C: RNA polymerase concentration (RNA polymerase/mass), rRNA and tRNA concentration (RNA/mass), RNA polymerase activity (fraction of total RNA polymerase actively engaged in RNA chain elongation), and stable RNA synthesis relative to total RNA synthesis. The following parameters increased 3.4-fold over the same temperature range: rRNA chain elongation rate, guanosine tetraphosphate (ppGpp) concentration, and culture growth rate. Above 40 degrees C, the changes became more complex, and the growth rate began to decrease. The observation that most RNA synthesis parameters are temperature invariant despite the increase of ppGpp suggests that the mechanism of RNA synthesis control by ppGpp, assumed to involve an interaction of RNA polymerase wtih ppGpp, is itself temperature dependent such that, with increasing temperature, higher concentrations of ppGpp are required to affect the RNA polymerase.

Bacterial Proteins↗
PubMed2004

Detection of mRNA degradation intermediates in tissues using the 3'-end poly(A)-tailing polymerase chain reaction method.

It has become increasingly clear that mRNA stability is an important determinant of mRNA abundance in virtually all organisms. Although our understanding of prokaryotic lower eukaryotic mRNA stability mechanisms has progressed considerably, little is known about mammalian mRNA stability mechanisms, particularly at the tissue and animal levels. This is due largely to the lack of suitable methods to approach the problem. In this study, we have developed and refined the 3'-end poly(A)-tailing polymerase chain reaction (PCR) method to detect degradation intermediates in vivo. Using an in vitro transcribed RNA as a template, we found that the method could be used to detect a homogeneous pool of RNA down to 0.1 ng. The addition of 10 microg of total RNA from tissues decreased the sensitivity limit to 4 ng. Detection limits of the technique were determined precisely by varying the concentrations of in vitro transcribed RNA in a constant amount of total RNA and varying the concentration of total RNA while maintaining a constant amount of in vitro transcribed RNA. Our overall results showed that the poly(A)-tailing PCR method could be used to detect specific RNA species of approximately 1000 nt in a pool of heterogeneous RNA in the range of 1 in 2500 to 1 in 10,000. To our knowledge, this is the most sensitive method to date for identifying mRNA degradation intermediates. Employing sense strand gene-specific primers in this method, we have discovered the class II and class III P-glycoprotein (Pgp) mRNA degradation intermediates in normal rat tissues. This method should serve as an additional tool to help us understand mRNA decay mechanisms in tissues and at animal levels.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2002

Optimization of RNA isolation from human ocular tissues and analysis of prostanoid receptor mRNA expression using RT-PCR.

PURPOSE: The isolation and analysis of human ocular RNA is problematic due to variables such as rapid degradation, tissue composition, and melanin contamination. The purpose of this work was to optimize an extraction protocol for the isolation of intact total RNA from a variety of diverse human ocular tissues and to employ RT-PCR to assess the expression of mRNA coding for all eight prostanoid receptors. METHODS: Total RNA was extracted from human iris, ciliary body, choroid, and retina using an RNeasy(R) Midi Kit. Total RNA was extracted from human cornea, sclera, and optic nerve using Tri-Pure(R) Isolation Reagent. 1.0 microgram of total RNA was reverse transcribed into cDNA and subsequently amplified by PCR (35 cycles) using primers designed against each of the human prostanoid receptor cDNAs. PCR products were analyzed by gel electrophoresis and endonuclease digestion. RESULTS: The total yield and quality of RNA derived from each tissue varied according to tissue composition and the isolation method employed. RT-PCR analysis revealed that each tissue expressed all prostanoid receptor mRNAs, however, 50 cycles of PCR was required to visualize FP receptor expression in scleral tissue. In all cases, prostanoid receptor mRNA expression was significantly lower than in human nonpregnant myometrium, which was used as the positive control. CONCLUSIONS: The different cellular composition of each ocular tissue ultimately dictated the methodology to be employed for the isolation of total RNA. Thus, two extraction protocols were optimized for the isolation of intact high quality RNA from a variety of human ocular tissues. The identification of all prostanoid receptor mRNAs in a diverse set of human ocular tissues suggests potential mechanisms for prostanoid-based therapeutics aimed at IOP reduction and stimulates speculation as to additional physiological and or pathophysiological roles mediated by prostanoids.

Choroid↗
PubMed1998

Granulocyte-colony stimulating factor inhibits TNFalpha production in a human hepatoma cell line.

The syndrome of cachexia associated with malignant diseases can be in part attributed to the effects of tumour necrosis factor alpha (TNFalpha) which itself is produced by a variety of tumour cells. We have recently reported that the human hepatoma cell line HepG2 expresses the TNFalpha gene and releases biologically active TNFalpha protein after stimulation with interleukin-1beta (IL-1beta). Granulocyte-colony stimulating factor (G-CSF) is a glycoprotein necessary for the proliferation and differentiation of neutrophil progenitor cells in the bone marrow. In addition G-CSF has been reported to exert anti-inflammatory effects. In our study we tested the effect of recombinant human G-CSF (rhG-CSF) on TNFalpha production in HepG2 cells. It could be shown that rhG-CSF (250 U/ml) significantly reduced IL-1beta-induced (300 pg/ml) TNFalpha gene expression after 1-h and 3-h incubation periods (TNFalpha mRNA concentrations were: 8.8+/-2.1 amol/ microg total RNA after a 1-h incubation with IL-1beta versus 3.8+/-1.3 amol/ microg total RNA after a 1-h incubation with IL-1beta + rhG-CSF and 13.8+/-2.2 amol/ microg total RNA after a 3-h incubation with IL-1beta versus 8.8+/-2. 1 amol/ microg total RNA after a 3-h incubation with IL-1beta + rhG-CSF). From these data we conclude that rhG-CSF is a potent inhibitor of cytokine-induced TNFalpha production by tumour cells. Therefore, treatment of patients with malignant diseases with rhG-CSF might represent a useful tool to improve the tumour-associated cachexia.

Carcinoma, Hepatocellular↗
PubMed1994

Expression of plasminogen activator inhibitor-1 mRNA in healthy, atherosclerotic and thrombotic human arteries and veins.

Plasminogen activator inhibitor-1 (PAI-1), specific inhibitor of plasminogen activators (PA), plays an important role in the regulation of fibrinolysis. Increased levels of PAI-1 have been associated with vascular disease such as thrombosis and atherosclerosis. In the present study the expression of PAI-1 mRNA in human healthy, atherosclerotic and thrombotic blood vessel walls was quantified by RNA-RNA hybridization in solution and localized by in situ hybridization. The mean expression of PAI-1 mRNA was significantly higher in healthy arteries (0.86 pg/microgram total RNA) than in healthy veins (0.29 pg/microgram total RNA), p < 0.01. The mean PAI-1 mRNA expression in thrombotic arteries (1.72 pg/micrograms total RNA) was significantly higher than that in healthy arteries, p < 0.05, and the mean PAI-1 mRNA expression in thrombotic veins (1.29 pg/micrograms total RNA) was significantly higher than that in healthy veins, p < 0.01. By in situ hybridization PAI-1 mRNA was detected in the intima, media and adventitia of healthy arteries and healthy veins. In atherosclerotic arteries PAI-1 mRNA was detected in the atherosclerotic plaque and in the medial and adventitial layers below the plaque. An increased expression of PAI-1 mRNA was found in the intimal layer of a thrombotic vein. The increased expression of PAI-1 mRNA in thrombotic arteries and veins indicates a role for PAI-1 in thrombogenesis.

Arteries↗
PubMed1985

Dictyostelium discoideum mRNAs developmentally regulated during spore germination have short half-lives.

mRNA decay was studied during spore germination in Dictyoselium discoideum by the use of three previously isolated cDNA clones, pLK109, pLK229, and pRK270, which are specific for mRNAs developmentally regulated during spore germination. The half-life of a constitutive mRNA, pLK125, which is present throughout germination, growth, and development, as also determined. Nogalamycin, a DNA-intercalating compound, was used to inhibit RNA synthesis. Total RNA was isolated at intervals after addition of the drug, and the decay of mRNAs specific for the cDNA clones was determined by both Northern blot and RNA dot hybridization. If nogalamycin was added immediately after activation of dormant spores, neither pLK229 nor pLK109 mRNA decayed, but pLK125 mRNA did decay. Although pLK109 mRNA did not decay under these conditions, the RNA was smaller 1 h after activation than in dormant spores, indicating that it was processed normally. At 1 h after activation, pLK229-, pLK125-specific mRNAs decayed exponentially, with half-lives of 24, 39, and 165 min, respectively. Under the same conditions, decay of pLK109-specific mRNA was biphasic. Thirty-eight percent of the mRNA decayed with a half-life of 5.5 min, and the remainder decayed with a half-life of 115 min. It seems likely that nogalamycin inhibits the synthesis of an unstable component of the mRNA degradative pathway which is needed continuously for the decay of pLK109 mRNA. By extrapolating the curve representing the rapidly decaying component, a half-life of 18 min was calculated for pLK109-specific mRNA. The mRNAs developmentally regulated during spore germination have half-lives shorter than that of the constitutive messenger and shorter than the average half-life of 3 to 4 h previously determined for total Dicyostelium polyadenylated mRNA.

Dictyostelium↗
PubMed1990

Simultaneous isolation of total cellular RNA and DNA from tissue culture cells using phenol and lithium chloride.

A rapid procedure for the isolation of intact total cellular RNA from cultured cells is described. This method combines the simultaneous disruption of cells and extraction of nucleic acids in a single step with the use of phenol and a buffer containing 100 mM LiCl. Total cellular RNA can be isolated in approximately 2 hours. The yield and quality of the RNA is comparable to the more widely employed methods requiring extensive preparatory steps such as extraction using guanidinium thiocyanate and subsequent CsCl gradient centrifugation. The RNA isolated using our procedure contains transcripts up to 10 kb in length and is suitable for Northern analysis. This procedure also yields high-molecular-weight DNA, which is a suitable substrate for restriction endonucleases.

Adrenal Gland Neoplasms↗
PubMed2001

Sterol regulatory element binding protein 1c (SREBP-1c) expression in human obesity.

OBJECTIVE: Investigation of the expression of sterol regulatory element binding protein-1c (SREBP-1c) in different adipose tissue depots in morbidly obese subjects before and after 1 year of weight loss induced by gastric banding operation. RESEARCH METHODS AND PROCEDURES: SREBP-1c expression was studied in 20 massively obese subjects (6 men and 14 women; age: 41 +/- 9 years; weight: 148 +/- 34 kg; percentage of body fat: 42 +/- 4; mean +/- SD) using reverse transcription competitive polymerase chain reaction. Adipose tissue biopsies were taken from omental, subcutaneous abdominal, and femoral depots before weight loss, and from subcutaneous depots after weight loss. Subcutaneous samples were taken also from 6 normal weight subjects. RESULTS: The level of SREBP-1c mRNA was significantly lower in omental (1.8 +/- 0.2 amol/microg of total RNA) than in subcutaneous abdominal (3.7 +/- 0.4 amol/microg of total RNA) or femoral (3.9 +/- 0.4 amol/microg of total RNA; p < 0.001, mean +/- SEM) depots. The values in subcutaneous depots were about twice as high in normal weight (7.4 +/- 2.5 for abdominal and 6.5 +/- 1.5 for femoral, p < 0.01) as in obese subjects. After weight loss, the mRNA levels of SREBP-1c increased in obese subjects, both in subcutaneous abdominal (5.3 +/- 0.7, p < 0.01) and in femoral (4.8 +/- 0.8, p < 0.05) tissue. DISCUSSION: SREBP-1c mRNA expression was lower in omental adipose tissue than in subcutaneous depots in obese subjects before weight loss. Furthermore, the expression of SREBP-1c in obese subjects was clearly lower than in normal weight subjects, but mRNA levels increased along with weight reduction. Weight reduction was associated with increased mRNA levels of SREBP-1c in obese subjects. The reduced expression of SREBP-1c in obesity could be ascribed to lowered action or concentration of insulin, changeable along with weight reduction. However, changes in SREBP-1c expression after weight reduction could also be ascribed to the changes in calorie intake or nutritional habits after gastric banding operation.

Abdomen↗