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At least 397 records · Page 22Linked to original sources
PubMed1998

Development of an in vivo method to study bovine intestinal response to dietary manipulation at the molecular level.

Our objective was to develop an in vivo method for analysis of nutrient-gene interactions in dairy cows at the molecular level. A series of techniques was applied to study the molecular response of cells in bovine small intestine to dietary manipulation. Three Holstein cows fitted with ruminal and duodenal cannulae were used for the investigation. As part of the validation of the procedure, lipid flow to the small intestine was manipulated using an abomasal infusion technique to study the expression of the gene encoding for the gastrointestinal hormone cholecystokinin (CCK). Biopsies (10 to 25 mg) were collected from the duodenum approximately 40 to 50 cm distal from the pyloric sphincter using a fiberoptic endoscope and biopsy forceps. Total RNA was isolated from the intestinal biopsies with yields of 20 to 50 microg. Cholecystokinin gene expression was analyzed in intestinal mucosa obtained from Holstein cows after slaughter either at a local abattoir or at our research facilities and compared with CCK gene expression in the biopsy samples collected from the cannulated animals. Using the technique of northern hybridization, we found that 15 microg of total RNA was sufficient to detect a strong CCK mRNA signal in the biopsy tissue samples, whereas 30 microg of total RNA showed a slightly weaker CCK mRNA signal in the postmortem duodenal tissues collected at our research facility. In contrast, the CCK mRNA signal in postmortem duodenal tissues obtained from cows slaughtered at the local abattoir was only barely detectable with 40 microg of total RNA. We conclude that the biopsy procedure is relatively noninvasive and is useful for monitoring intestinal responses to dietary manipulation and to physiological changes, such as those that occur during lactation and with metabolic disorders. The application of the method will provide insight into nutrient-gene interactions that occur in bovine intestinal mucosa.

Abattoirs↗
PubMed2001

Noninvasive diagnosis of renal-allograft rejection by measurement of messenger RNA for perforin and granzyme B in urine.

BACKGROUND: Acute rejection is a serious and frequent complication of renal transplantation, and its diagnosis is contingent on the invasive procedure of allograft biopsy. A noninvasive diagnostic test for rejection could improve the outcome of transplantation. METHODS: We obtained 24 urine specimens from 22 renal-allograft recipients with a biopsy-confirmed episode of acute rejection and 127 samples from 63 recipients without evidence of acute rejection. RNA was isolated from the urinary cells. Messenger RNA (mRNA) encoding the cytotoxic proteins perforin and granzyme B and a constitutively expressed cyclophilin B gene were measured with the use of a competitive, quantitative polymerase chain reaction, and the level of expression was correlated with allograft status. RESULTS: The log-transformed mean (+/-SE) levels of perforin mRNA and granzyme B mRNA, which encode cytotoxic proteins, but not the levels of constitutively expressed cyclophiiin B mRNA, were higher in the urinary cells from the 22 patients with a biopsy-confirmed episode of acute rejection than in the 63 recipients without an episode of acute rejection (perforin, 1.4+/-0.3 vs. -0.6+/-0.2 fg per microgram of total RNA; P<0.001; and granzyme B, 1.2+/-0.3 vs. -0.9+/-0.2 fg per microgram of total RNA; P<0.001). Analysis involving the receiver-operating-characteristic curve demonstrated that acute rejection can be predicted with a sensitivity of 83 percent and a specificity of 83 percent with the use of a cutoff value of 0.9 fg of perforin mRNA per microgram of total RNA, and with a sensitivity of 79 percent and a specificity of 77 percent with the use of a cutoff value of 0.4 fg of granzyme B mRNA per microgram of total RNA. Sequential urine samples were obtained from 37 patients during the first nine days after transplantation; and measurements of the levels of mRNA that encoded cytotoxic proteins identified those in whom acute rejection developed. CONCLUSIONS: Measurement of mRNA encoding cytotoxic proteins in urinary cells offers a noninvasive means of diagnosing acute rejection of renal allografts.

Acute Disease↗
PubMed2004

Gold nanoparticle probe-based gene expression analysis with unamplified total human RNA.

Microarray-based gene expression analysis plays a pivotal role in modern biology and is poised to enter the field of molecular diagnostics. Current microarray-based gene expression systems typically require enzymatic conversion of mRNA into labeled cDNA or cRNA. Conversion to cRNA involves a target amplification step that overcomes the low sensitivity associated with commonly used fluorescent detection methods. Herein, we present a novel enzyme-free, microarray-based gene expression system that uses unamplified total human RNA sample as the target nucleic acid. The detection of microarray-bound RNA molecules is accomplished by targeting the poly-A tail with an oligo-dT20 modified gold nanoparticle probe, signal amplification by autometallography, and subsequent measurement of nanoparticle-mediated light scattering. The high sensitivity afforded by the nanoparticle probes allows differential gene expression from as little as 0.5 microg unamplified total human RNA in a 2 h hybridization without the need for elaborate sample labeling steps.

Biosensing Techniques↗
PubMed2002

Quantitative real-time reverse transcription polymerase chain reaction: normalization to rRNA or single housekeeping genes is inappropriate for human tissue biopsies.

Careful normalization is essential when using quantitative reverse transcription polymerase chain reaction assays to compare mRNA levels between biopsies from different individuals or cells undergoing different treatment. Generally this involves the use of internal controls, such as mRNA specified by a housekeeping gene, ribosomal RNA (rRNA), or accurately quantitated total RNA. The aim of this study was to compare these methods and determine which one can provide the most accurate and biologically relevant quantitative results. Our results show significant variation in the expression levels of 10 commonly used housekeeping genes and 18S rRNA, both between individuals and between biopsies taken from the same patient. Furthermore, in 23 breast cancers samples mRNA and protein levels of a regulated gene, vascular endothelial growth factor (VEGF), correlated only when normalized to total RNA, as did microvessel density. Finally, mRNA levels of VEGF and the most popular housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were significantly correlated in the colon. Our results suggest that the use of internal standards comprising single housekeeping genes or rRNA is inappropriate for studies involving tissue biopsies.

Biopsy↗
PubMed2001

Increments in insulin sensitivity during intensive treatment are closely correlated with decrements in glucocorticoid receptor mRNA in skeletal muscle from patients with Type II diabetes.

To test the hypothesis that changes in the expression of the glucocorticoid receptor (GCR) and the beta(2)-adrenoceptor (beta(2)-AR) contribute significantly to the abnormal glucose metabolism in skeletal muscle from patients with Type II diabetes, we have examined (1) the levels of total GCR (alpha+beta isoforms), the alpha/alpha 2 isoform of GCR and beta(2)-AR mRNAs in skeletal muscle from insulin-resistant patients with Type II diabetes (n=10) and healthy controls (n=15), and (2) the effects of 8 weeks of intensive treatment on the whole-body glucose disposal rate and on total GCR, alpha/alpha 2 GCR and beta(2)-AR mRNA levels in diabetic patients. The total glucose disposal rate was measured by the euglycaemic hyperinsulinaemic (2 m-units x min(-1) x kg(-1)) clamp technique, and mRNA levels were assessed by reverse transcriptase-PCR and HPLC for separation of standard and unknown and quantification. Mean levels of total GCR and alpha/alpha 2 GCR mRNAs were increased in patients with Type II diabetes when compared with control subjects [total GCR, 2.06+/-0.30 and 1.47+/-0.10 amol/microg of total RNA respectively (P=0.09); alpha/alpha 2 GCR mRNA, 1.69+/-0.31 and 0.92+/-0.09 amol/microg of total RNA respectively (P=0.02)], whereas mRNA levels of the beta isoform of GCR (total GCR minus alpha/alpha 2 GCR) were decreased (P=0.006). beta(2)-AR mRNA levels were comparable in diabetic patients and control subjects (0.53+/-0.05 and 0.45+/-0.02 amol/microg of total RNA respectively; P=0.2). Intensive treatment for 8 weeks was associated with improved glycaemic control (P=0.019), and during the clamp a 75% (P=0.001) increase in the whole-body insulin-stimulated glucose disposal rate was demonstrated. Total GCR (P=0.005), alpha/alpha 2 GCR (P=0.005) and beta(2)-AR (P=0.03) mRNA levels all decreased significantly after intensive insulin treatment. A close correlation was found between increments in glucose uptake during intensive treatment and decrements in skeletal muscle total GCR mRNA (r=0.95, P<0.001; multiple regression analysis), and between glucose uptake and alpha/alpha 2 GCR m RNA levels (r=0.88, P<0.001; simple correlation). In conclusion, the abnormal regulation of GCR mRNA is likely to play a significant role in the insulin resistance observed in obese patients with Type II diabetes.

Adult↗
PubMed1980

[Potential oncogenic action of the RNA isolated from rat lymphosarcoma].

From the tissue of the transplantable strain of rat lymphosarcoma, using phenol technics, a total RNA (sedimentation constants 31,5S; 19,0S; 6S) and chromosome-nuclear mRNA were isolated. The preparations contained up to 10% of protein. Nucleic acids (in a dosage of 10 mg RNA) were given by single intraperitoneal injections to 2 month-old rats. The animals showed a 180% increase of the lymphatic neoplasms yield when injected both total RNA and mRNA. Transplantation into newborn rats of the rat lymphocytes, treated in vitro by the total RNA preparations, failed to yield such a significant increase in the occurrence of neoplasms. A molecular weight of RNA fragments was similar to that in the experiments on transfection by nucleic acids, isolated from tumors of viral etiology.

3,3'-Dichlorobenzidine↗
PubMed1978

[Isolation and characterization of beef thyroid giant RNA].

Total HMW-RNAs were prepared by three different methods (method with phenol, method with NaClO4, method without phenol using Ultrogel AcA 22 filtration). Giant RNAs were obtained in the void volume by filtration on Sepharose 2B. The giant RNAs/total HMW-RNA ratio is higher (6.77%) with the gel filtration method than with phenol or NaClO4 methods (1.41% and 1.00% respectively). The nucleotide composition of these RNAs is DNA-like and the sedimentation constants are approximately 70-100 S.

Animals↗
PubMed2004

[Gene differential expression of liver tissues in crossbred versus purebred chicken and their relationship with heterosis of meat trait].

The concept of heterosis has already been put forward for a century. The hypothesis of Dominance, Superdominace and Epistasis has also been brought forward to explain the phenomenon of heterosis. As we know, there is spatio-temporal speciality about the expression of gene and only expressed genes contribute to the formation of heterosis. So the study on heterosis in expression level becomes more meaningful. A lot of studies on heterosis in this level have been done in plants, but there is no such study carried on animals in this area. In this study, the technique of mRNA Reverse Transcription Differential Display was used to research the heterosis molecular mechanism of animal. In order to expound the molecular genetic mechanism of animals heterosis, the 4 x 4 completely diallele cross experiment of 4 purebreds chicken was conducted among White Polymouth Rock (EE), Chinese Silk Chicken (CC), CAU Brown (DD) and White Leghorn (AA). The chicken of 16 cross combinations were reared to 8 weeks old, then 30 chicken in each combination were selected randomly and slaughtered. The traits of body weight of 8 weeks, wing weight, eviscerated weight, eviscerated weight with giblet, breast muscle yield, leg muscle yield, body length, abdomen fat weight, intramuscular fat width, tibia length were measured, and in which 8 individuals in each combination were selected randomly to collect the liver tissue samples, which were stored in liquid nitrogen or at -80 degrees C to be used for total RNA (TRNA) extracting. After the total RNA (TRNA) was extracted, 16 TRNA pools were formed in the same quantitative according to the concentration of 8 individual TRNA. They were reversely transcribed with three anchor primers H-T11 A, H-T11 G and H-T11 C. Then the reverse transcription PCR for each transcript product was done in two repeats at the same time with the same anchor primers and 8 random primers. The polyacrylamide gel electrophoresis of each PCR product was run in Bio-Rad Power 3,000 temperature control system. After electrophoresis, the gel was stained by AgNO3 according to the stain method described by Echt et al. The differential display bands in the polyacrylamide gel were counted. The band displayed is counted as 1 whereas no band is counted as 0 and only expression bands reproducible in two repeats were statistically analyzed. The correlation analysis between heterosis percentage and gene expression patterns was done with statistic analysis software (SAS) package. The statistic results indicated that among 690 total numbers of bands, the percentage of differential expression bands reproducible (457) is 66.23%. Eight kinds of gene differential expression patterns were found and listed as follow: 1): Band presents only in one purebred (P1);2): Band in one crossbred and its corresponding paternal purebred; or Band in one crossbred and its corresponding maternal purebred (P2);3): Band in purebreds and one crossbred (P3); 4): Band only in one crossbred (P4);5): Bands in both crossbreds and one purebred (P5);6): Bands only in both crossbreds (P6);7): Bands only in purebreds (P7);8): Bands both in purebreds and crossbreds P8. The differential expression of gene between purebred and crossbred chicken was detected for the first time. The proportion of each pattern in each kind of purebred combination is different. The percentage of P8 (75.34%) is the highest. The total percentage of differential expression patterns (24.66%) showed that the gene differential expression exists as a matter of fact. Among all the gene differential expression patterns, the percentage of P3 is the highest whereas the percentage of P7, P6 and P4 is very low, it indicated that different genes may have different expression patterns in purebreds and crossbreds. The results are similar to the study results on plants, which indicates that the gene differential expression between purebred and crossbred exists universally in biology. The correlation between gene expression patterns and heterosis percentage was studied, but correlation between P8 and the heterosis percentage is not significant (P > 0.05), it indicates that some patterns of gene differential expression may be the molecular genetic basic of heterosis. Among all the gene differential expression patterns, each pattern affects the expression of meat trait in different manner. There is significantly negative correlation between P4 and heterosis percentage of body weight of 8 weeks, breast muscle yield, leg muscle yield, eviscerated weight with giblet and eviscerated weight (P < 0.05); P1 is of significantly negative correlation with heterosis percentage of abdomen fat weight (P < 0.05) and of very significantly negative correlation with heterosis percentage of body length (P < 0.01); The negative correlation between P2 and heterosis percentage of intramuscular fat width is significant (P < 0.05); The positive correlation between P7 and heterosis percentage of leg muscle yield, wing weight, eviscerated weight with giblet and intramuscular fat width is significant (P < 0.05); The positive correlation between P5 and heterosis percentage of tibia length (P < 0.05) is significant. These results show that these 5 kinds of patterns play important role in heterosis forming of meat trait. P1 and P7 show that expressed gene in purebreds is depressed; P4 indicates that new gene expression occurs in crossbreds; P5 reveals that expressed gene only in one purebred express in all crossbreds. All genes of crossbreds come from purebred, which are not only the simple adding of these purebred genes, giving birth to unknown interaction between these genes coming from different purebreds, then leading to differential expression of genes. These gene differential expressions maybe form the heterosis of meat trait.

Animals↗
PubMed2004

Cross-species hybridization: characterization of gene expression in woodchuck liver using human membrane arrays.

Total RNA from normal adult woodchucks was analyzed using membrane arrays containing human cDNA clones, and the gene expression patterns were compared to human liver. Various hybridization and wash conditions were examined. In both the woodchuck and human livers, 352 genes were identified as highly expressed (Z-scores > or =1.96). These genes represented numerous liver functions: transcription, RNA processing, signal transduction, protein synthesis and degradation, as well as enzymes. Several genes were selected and expression was verified by Northern blots for woodchuck liver. There were no false positives but 29 genes were identified as false negatives, expressed only in human liver. Possible reasons for these false negatives were the length and percentage of homology between the two species, differences in the distribution and types of mismatches, and the sequence region spotted on the array. These were assessed by examining expression of the transferrin gene in both species. A 200-fold range of RNA concentration (0.1-20 microg total RNA) was also examined and the optimal RNA concentration was determined to be 5 microg. Membranes were capable of being hybridized and reprobed at least five times. The study demonstrates that cross-species hybridization is a valid method for identifying gene expression in woodchuck liver.

Animals↗
PubMed2004

Hormone and metabolic factors associated with leptin mRNA expression in pre- and postmenopausal women.

Recent information has extended leptin's action, beyond the control of appetite, to various sites of metabolic regulation. To better understand leptin's role we studied its production in subcutaneous and visceral fat compartments before and after menopause. During elective abdominal surgery, biopsies of subcutaneous and omental tissues were taken from 20 women at pre- (BMI 28.4 +/- 4.5 kg/m2) and 10 at postmenopause (BMI 30.6 +/- 7.7 kg/m2). In both groups serum leptin levels were similar, and highly correlated with BMI. In subcutaneous adipose tissue, leptin mRNA expression was significantly higher in pre- than in postmenopausal women (50.4 +/- 20.5 amol/microg total RNA versus 34.5 +/- 24.9 amol/microg total RNA, respectively). Leptin mRNA expression in subcutaneous tissue was independently correlated with fasting glucose (R = 0.89, P < 0.006) at premenopause, and with serum estradiol (R = 0.77, P < 0.04) at postmenopause. Leptin mRNA expression in visceral fat was correlated with DHEAS (R = 0.86, P < 0.001), at premenopause. These results indicate that in both compartments, leptin production is sensitive to different but overlapping stimuli, conveying information about energy availability to central and peripheral sites under different conditions of estrogen exposure.

Adipose Tissue↗
PubMed1997

Sequence and tissue distribution of chicken cellular nucleic acid binding protein cDNA.

We sequenced cDNAs coding for chicken cellular nucleic acid binding protein (CNBP). Two slightly different variations of the open reading frame were found, each of which translates into a protein with seven zinc finger domains. The longest transcript contains an in-frame insert of 3 bp. The sequence conservation between chick CNBP cDNAs with human, rat and mouse CNBP cDNAs is extreme, especially in the coding region, where the deduced amino acid sequence identity with human, rat and mouse CNBP is 99%. CNBP-like transcripts were also found in various tissues from insect, shrimp, fish and lizard. Regions with remarkable nucleotide conservation were also found in the 3' untranslated region, indicating important functions for these regions. Quantitative reverse transcription polymerase chain reaction (RT-PCR) indicated that in the chick, CNBP in present in all tissues examined in approximately equal ratios to total RNA. RT-PCR of total RNA isolated from different phyla indicate CNBP-like proteins are widespread throughout the animal kingdom. The extraordinary level of conservation suggests an important physiological role for CNBP.

Amino Acid Sequence↗
PubMed2000

Lymphocyte glucocorticoid receptor mRNA correlates negatively to serum leptin in normal weight subjects.

OBJECTIVE: The aim of the present study was to test the hypothesis that glucocorticoid receptor mRNA concentrations decreased with increasing fatness in normal subjects. MEASUREMENTS: Serum leptin concentrations, fat distribution parameters, lymphocyte glucocorticoid (GCR) mRNA, beta2-adrenoceptor mRNA and c-fos mRNA concentrations measured by RT-PCR-HPLC. SUBJECTS: Fifteen healthy non-obese young subjects with a mean body mass index (BMI) of 23. 5+/-0.3 (+/-s.e.m.) kg/m2. RESULTS: Lymphocyte GCR and beta2-adrenoceptor mRNA concentrations averaged 4.2+/-0.2 (+/-s.e.m. ) amol/microg total RNA and 1.4+/-0.1 amol/microg total RNA, respectively. There was a significant negative correlation between serum leptin and lymphocyte GCR mRNA (P<0.01). Serum leptin correlated positively with the waist-hip ratio (P<0.03), whereas lymphocyte GCR mRNA correlated negatively to the waist-hip ratio (P<0.04). Serum cortisol correlated with the weight of the subjects but not the waist-hip ratio or GCR mRNA. CONCLUSIONS: It is suggested that the decrease in lymphocyte GCR mRNA concentration with increasing serum leptin concentrations is a counterregulatory response to an increased body fat content. Further studies are warranted, especially to elucidate the relationship between GCR mRNA in lymphocytes and in fat cells and to clarify the mechanism of the decrease in GCR mRNA.

Adipocytes↗
PubMed1991

Molecular cloning of a heart antigen that cross-reacts with a neutralizing antibody to Coxsackievirus B4.

A panel of coxsackievirus B4 (CVB4) neutralizing monoclonal antibodies was tested against a panel of normal mouse tissues. One mAb, 356-1, reacted specifically with murine heart tissue. Examination of the reactivity of 356-1 with CVB4 polypeptides using Western immunoblotting revealed that 356-1 binds to the VP-1 capsid protein. Immunohistochemical studies revealed an A band pattern of staining of the heart by this antibody. Western immunoblotting of sequential differential extracts of heart showed that 356-1 predominantly reacted with the murine cardiac myosin heavy chain. A rather weak cross-reaction was found with actin. These observations were confirmed by the binding of 356-1 to purified cardiac myosin and actin. This antibody showed a higher affinity for murine cardiac muscle myosin than for skeletal muscle myosin. The monoclonal antibody 356-1 was then used to screen a lambda gt11 CD1 mouse heart cDNA expression library. Forty-eight positive plaques were obtained, 14 of which reacted strongly with the antibody and were selected for additional studies. In 13/14 clones, inserts were amplified using the polymerase chain reaction (PCR). The PCR products ranged in size from approximately 150 to 1400 bp. Northern hybridization using these inserts demonstrated that 10/13 recognized a approximately 6.5 kb message in mouse heart total RNA and not in liver total RNA. These amplified inserts were sequenced and were found to contain sequences which encode approximately 1/3 of the amino-terminal end of light meromyosin. By comparison with known sequences of rat alpha and beta cardiac myosin heavy chains, we were able to identify sequences specific for alpha chain as potential cross-reactive autoantigenic epitope.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Regulation of the apolipoprotein AIV gene expression by estrogen differs in rat and mouse.

Previously we have shown that estrogen administration to Sprague Dawley rats and to the inbred C3H/HeJ mouse strain produced different effects on plasma lipoproteins [Srivastava, R. A. K., Baumann, D. & Schonfeld, G. (1993) Eur. J. Biochem. 216, 527-538]. While low-density lipoprotein (LDL) levels fell in rats, they rose in mice. Plasma apoprotein (apo) AI levels and high-density lipoprotein (HDL) cholesterol fell in both species but by much less in mice than in rats. Since apolipoproteins AIV and AII are two other protein constituents of HDL, we wished to test the hypothesis that estrogen would produce different effects on these apoproteins in mice and rats. Male rats and C3H/HeJ mice were administered 17 beta-estradiol at 5 micrograms.g body mass-1.day-1 for six consecutive days. In a separate experiment, castrated male C3H/HeJ mice were administered beta-estradiol [(0.16 micrograms.g body mass-1.day-1 or 5.0 micrograms.g body mass-1.day-1, or testosterone (1 microgram/g)] for 14 days. ApoAIV mRNA levels were determined in total liver, in liver nuclei and in total intestine. Rat hepatic apoAIV mRNA decreased twofold (from 16.5 +/- 3 pg/micrograms total RNA to 7.1 +/- 2.5 pg/micrograms total RNA) while mouse hepatic and nuclear apoAIV mRNA both increased 1.5-2-fold. Intestinal apoAIV mRNA decreased in mice and increased in rats. Testosterone had no effects. Nuclear apoAIV mRNA transcription rates in rat and mouse liver changed little, if at all, indicating that estrogen-induced changes in steady-state levels of apoAIV mRNA were not determined by hepatic transcriptional mechanisms. Both species possessed similar apoAIV mRNA transcription start sites. To assess whether other mouse strains also differed from rats, we surveyed 13 other inbred mouse strains. Some strains increased hepatic apoAIV mRNA, some did not change but, in contrast to rat, no strain experienced a fall in mRNA levels. Estrogen-induced changes in plasma apoAIV levels were not correlated with changes in the levels of hepatic apoAIV mRNA levels. These data indicate that (a) apoAIV mRNA levels are regulated differently by estrogen in mouse and rat livers and intestines, (b) regulation of apoAIV mRNA by estrogen is both mouse strain and tissue specific and (c) regulation of plasma apoAIV is achieved by mechanisms other than those depending on the steady-state levels of hepatic apoAIV mRNA. In contrast with apoAIV mRNA, estrogen decreased hepatic apoAII mRNA both in rat (threefold) and in mouse (twofold) and parallel changes were observed in transcription rates.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2005

[Differentially expressed gene profiles of cochlea in rats induced by acute and chronic sodium salicylate injection].

OBJECTIVE: To study the mechanism of electrophysiologic changes caused by different type of sodium salicylate injection. METHODS: Decapitated three group rats ( acute injected, chronic injected and normal rats ) separately, dissected the temporal bones to collect cochlea, and the otic capsules were removed. Then the cochlear materials from each groups were pooled and homogenized respectively, extracted the total RNA, obtained cDNA from purified total RNA by reversed transcription, cDNA were transcripted to cRNA probes in vitro. Hybridized the cRNA probes with tester chip to evaluate the quality of probes, if good, hybridized the probes with real chip. Obtained three gene expression profiles of different groups of cochlea Analyzed the differentially expressed genes among three groups by SOM. Analogized the SOM result to electrophysiologic changes. Then analyzed the genes in clusters of analog results by Gene Ontology. Then the genes in clusters of analog results were analyzed by Gene Ontology. Hsp27 was chosen to validate the result of gene chip using real time quantitative reverse transcription PCR ( RTQ RT-PCR). RESULTS: The probes was good, and the chip hybridization results was credible. We obtained 6 clusters genes by SOM analysis, in which we choose cluster 3 and cluster 4 as candidate cluster. There were 46 genes in cluster 3 and 30 genes in cluster 4 employing GO analysis, which involved in cell communication, cell motility, metabolism, immune response and nerve ensheathment, et al. The result of RTQ RT-PCR showed high concordance with that of gene chip. CONCLUSION: It's a new method to study the mechanism of electrophysiologic changes caused by sodium salicylate by gene chip and SOM analysis.

Animals↗
PubMed2004

Use of RNA amplification in the optimal characterization of global gene expression using cDNA microarrays.

Microarray analysis of human tissue is frequently hindered by the limited amount of RNA available. Although amplification protocols can be utilized, the relative representation of transcripts present in the starting material must remain unaltered. In this study, 200 ng of total RNA derived from cultured renal epithelial cells from tuberous sclerosis complex (TSC) carriers and control individuals was amplified by in vitro transcription with T7 RNA polymerase. The resulting Cy-labeled cDNAs (from total or amplified RNA (aRNA)) were analyzed as direct replicates and dye-flips on slides containing 10,000 human cDNAs. The Pearson correlation coefficients for the direct replicate experiments were 0.80 (20 microg total RNA), 0.85 (40 microg total RNA), and 0.93 (2 microg of aRNA). Comparisons between the array data revealed that the majority of genes expressed in total RNA (97% for 20 microg and 85% for 40 microg) were also detected in aRNA. The correlation coefficient of the expression ratios for genes detected in both total RNA (40 microg) and aRNA was 0.63. Further, Student's t-test indicated no significant difference (P = 0.83) between these ratios. These results indicate that the number of expressed genes detected with total RNA is proportional to the amount of RNA used and underscore the requirement of large amounts of total RNA for a comprehensive characterization of gene expression profiles. RNA amplification allows the detection of a large number of genes expressed in the starting RNA population without altering their relative intensities significantly. Thus, an RNA amplification step improves the quality of gene expression results obtained by microarray analysis. This study indicates that high quality microarray data can be generated from small amounts of RNA, including those extracted from limiting clinical samples and microdissected histological specimens.

Gene Expression Profiling↗
PubMed2000

In-vitro cyclic tensile loading of an immobilized and mobilized ligament autograft selectively inhibits mRNA levels for collagenase (MMP-1).

To test the hypothesis that loading conditions can be used to "engineer" ligament autograft behaviors, the effect of cyclic tension on the mRNA levels of matrix molecules and collagenase in in-vivo immobilized and mobilized 6-week rabbit medial collateral ligament (MCL) autografts was examined using an in-vitro system. Femur-[autograft MCL]-tibia complexes were subjected to a tensile stress of 4 MPa at 0.5 Hz for 1 min, followed by 14 min of rest. This 15-min testing cycle was repeated for 4 h. Semi-quantitative reverse transcrip-tase polymerase chain reaction (RT-PCR) was performed on RNA from mechanically treated MCL autografts, using rabbit-specific primer sets for types I and III collagen, biglycan, decorin, fibromodulin, lumican, versican, matrix metalloproteinase-1 (MMP-1, collagenase-1), MMP-13 (collagenase-3), and a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Interestingly, 4 h of culture of normal control MCLs led to increased mRNA levels for MMP-1 (P < 0.05), but there were no significant changes in MMP-13 mRNA levels. Total RNA levels in that normal MCL tissue were, however, decreased after culture (P < 0.05). In-vitro tensile loading of in-vivo mobilized autografts resulted in a significant increase in total RNA (185% of in-vitro non-loaded autografts). On the other hand, in-vitro tensile loading of in-vivo immobilized autografts resulted in no significant changes in total RNA levels compared with levels in non-loaded control grafts. MMP-1 mRNA levels in both the in-vivo mobilized (47% of non-loaded autograft) and in-vivo immobilized (38% of non-loaded autograft) MCL autografts were significantly lower than those in non-loaded control tissue following in-vitro tensile loading, but there were no significant changes in the mRNA levels for the seven other matrix molecules assessed. These results show that it is possible to selectively inhibit MMP-1 mRNA levels in autograft ligaments by supplying mechanical stimuli in vitro. The results also demonstrate that in-vivo immobilization leads to a decrease in the effects of subsequent in-vitro mechanical loading in such autografts with respect to total RNA levels. Collectively, these results demonstrate that both in-vivo and in-vitro loading have implications in the engineering of an ideal ligament graft.

Animals↗
PubMed1993

Developmental expression of the alpha 6 GABAA receptor subunit mRNA occurs only after cerebellar granule cell migration.

Using a competitive polymerase chain reaction (PCR) and appropriate internal standards, we have analyzed absolute amounts of the alpha 6 GABAA receptor subunit mRNA in the postnatally developing cerebellum and neocortex. The PCR data have shown that absolute amounts of the alpha 6 receptor subunit mRNA in the cerebellum increase dramatically (nearly 100-fold) during the second postnatal week, reaching maximal levels by postnatal day 21 (1 fmol/microgram total RNA). The absolute amount of the alpha 6 GABAA receptor subunit mRNA in the cortex at postnatal day 1 was 2 amol/microgram total RNA and increased to 7 amol/micrograms total RNA by postnatal day 14. No further increase in alpha 6 mRNA expression in the adult cortex was observed. Microscopic analysis of emulsion coated and counterstained sections indicated that alpha 6 GABAA receptor subunit mRNA labeling was only detected in the internal granule cell layer and not in either the external granule cell layer or in migrating granule cells. The alpha 1 GABAA receptor subunit mRNA increased in the cerebellar cortex with a similar temporal profile, although its distribution extended to additional cell types (Purkinje cells, stellate/basket cells and possibly cerebellar astrocytes). The temporal expression of these two GABAA receptor subunit mRNAs is coincident with the formation of synaptic contacts in the granule cell dendrites suggesting that afferent pathways innervating these neurons following cell migration may play a critical role in increasing the expression of mRNAs encoding the alpha 1 and alpha 6 GABAA receptor subunits.

Aging↗