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At least 55 records · Page 3Linked to original sources
PubMed2002

Quantitative gene expression analysis in renal biopsies: a novel protocol for a high-throughput multicenter application.

BACKGROUND: Recent advances in gene expression analysis may add the quantification of mRNA species in renal biopsies to routine diagnostic procedures in nephrology. METHODS: A systematic evaluation was performed on the relevant steps required to efficiently obtain cDNA from renal biopsies for high-throughput reverse transcription-polymerase chain reaction (RT-PCR) based mRNA quantification. RESULTS: The protocol preserves mRNA integrity by a novel RNase inhibitor and allows meticulous microdissection followed by maximal RNA recovery from tissue samples. Reverse transcription was optimized to give the best yield from minimal starting material. RNA quantity and quality were systematically investigated by real-time RT-PCR and electrophoresis on a microfluidic system, respectively. The reported procedure offers high RNA preservation and increases the yield of cDNA significantly compared to former protocols. CONCLUSION: The simplicity of biopsy material acquisition combined with the centrally performed processing makes this protocol suitable for a wide spectrum of expression analysis in diverse clinical settings.

Biopsy↗
PubMed2003

PCR-based quantification of amplified RNA from laser microdissected mouse liver samples.

New molecular methods such as quantitative reverse transcription-polymerase chain reaction (RT-PCR) and microarray gene expression analysis have been established recently to quantify gene expression in tissue samples. Such methods, although highly sensitive, require RNA quantities of at least several micrograms. These amounts are not available in many experiments concerning microdissected embryonic or regenerating structures. We combined laser-assisted tissue preparation, RNA amplification, and quantitative RT-PCR to estimate both accuracy and linearity of gene expression in small tissue samples. Our results show that mRNA isolated from laser-microdissected fetal liver tissue or regenerative nodules, which originated from EGFP-marked transplanted fetal cells, can be significantly increased with the amplification protocol. The quantitative expression ratio of the genes albumin and GAPDH was conserved after one and two rounds of amplification compared to nonamplified material. Furthermore, genes expressed at low levels such as the transcription factor C/EBPbeta become detectable after two rounds of amplification in small microdissected tissue samples.

Albumins↗
PubMed2006

Options available for profiling small samples: a review of sample amplification technology when combined with microarray profiling.

The possibility of performing microarray analysis on limited material has been demonstrated in a number of publications. In this review we approach the technical aspects of mRNA amplification and several important implicit consequences, for both linear and exponential procedures. Amplification efficiencies clearly allow profiling of extremely small samples. The conservation of transcript abundance is the most important issue regarding the use of sample amplification in combination with microarray analysis, and this aspect has generally been found to be acceptable, although demonstrated to decrease in highly diluted samples. The fact that variability and discrepancies in microarray profiles increase with minute sample sizes has been clearly documented, but for many studies this does appear to have affected the biological conclusions. We suggest that this is due to the data analysis approach applied, and the consequence is the chance of presenting misleading results. We discuss the issue of amplification sensitivity limits in the light of reports on fidelity, published data from reviewed articles and data analysis approaches. These are important considerations to be reflected in the design of future studies and when evaluating biological conclusions from published microarray studies based on extremely low input RNA quantities.

Gene Expression Profiling↗
PubMedDate not supplied

[Participation of the genetic apparatus of the hippocampal and neocortical cells in the mechanisms of action of desglycine-arginine vasopressin on learning and memory processes in rats].

The paper deals with the influence of synthetic vasopressin analogue--desglycine-arginine vasopressin (DG-AVP) on the content of RNA and fractional composition of chromatin proteins in the tissue of neocortex and hippocampus of intact white rats and after establishing of two-way avoidance reflex. Administration of the peptide alone significantly increased RNA content in hippocampal tissue, injection of the peptide 10 min before conditioning did not lead to significant changes in RNA quantity as compared to that in animals in which the conditioned reflex was established against the background of saline administration. In neocortical tissue neither learning itself nor administration of DG-AVP alone was accompanied by significant changes in RNA content, while learning against the background of peptide injection significantly increased RNA in that structure. In hippocampal and neocortical tissues quantitative changes were observed in certain fractions of chromatin proteins in all animal groups studied.

Animals↗
PubMed2004

A fluid cover medium provides superior morphology and preserves RNA integrity in tissue sections for laser microdissection and pressure catapulting.

Laser microdissection and pressure catapulting has become a powerful tool to obtain homogeneous cell populations from tissue samples in nearly all fields of biomedical research. The isolated cells can be subsequently used for the analysis of proteins, DNA or RNA. However, the method requires physical access to the tissue surface and the sections therefore need to be air-dried and uncovered. The consequence is poor morphology, which severely reduces the potential of the technique, especially in non-homogeneous tissues or tissues with infiltrating immune cells. To overcome this limitation, a fluid cover medium was developed and the effects on frozen and paraffin wax-embedded tissue morphology were evaluated. The cover medium improved the morphology such that it was almost comparable to sections overlaid with glass coverslips. Moreover, the laser microdissection procedure was facilitated, since the medium allowed larger areas of tissues to be laser pressure-catapulted. Neither the isolation of proteins nor the extraction of genomic DNA was adversely affected by the use of the fluid cover medium. No significant differences in RNA quantity and integrity were detected by TaqMan real-time PCR for GAPDH, and microchip electrophoresis, between covered and uncovered tissue sections. In conclusion, this method provides considerably improved morphology for laser microdissection and pressure catapulting techniques without affecting RNA-dependent downstream applications. This not only facilitates established procedures, but will also extend the application to tissues that require superior morphological resolution.

Basal Cell Carcinoma↗
PubMed2006

Analysis of gene expression in mineralized skeletal tissues by laser capture microdissection and RT-PCR.

The analysis of gene expression by growth plate chondrocytes in vivo has been hampered by the inherent difficulty in performing in situ hybridization on mineralized tissues. The combination of laser capture microdissection and reverse transcription-polymerase chain reaction (RT-PCR) allows analysis of gene expression by cells selectively removed from histologic sections by laser ablation. In order to apply this method to mineralized tissues, a decalcification process is required. The object of this study was to determine the optimal method for tissue decalcification prior to laser capture microdissection RT-PCR that will preserve integrity of the mRNA population. Acetone, 10% formalin, and methacarn were evaluated as fixatives, while Surgipath Decalicifier I, 10% ethylenediaminetetraacetic acid (EDTA), and 20% EDTA were evaluated as decalcifying reagents. Our results demonstrate that the optimal RNA quality was preserved by a decalcification protocol consisting of 20% EDTA for decalcification followed by fixation in methacarn, although this method is also associated with a reduction in RNA quantity.

Animals↗
PubMed1978

[Changes in the RNA concentration in neurons of the superior olivary complex in cats following monaural and binaural stimulation].

The nature of binaurally converging connections on the level of the superior olive nuclei was studied in cats anesthetized by the method of quantitative cytospectrophotometry. Significant increase in cytoplasmic RNA content in neurons of ipsi- and contralateral medial and lateral nuclei was observed after 2 hrs monaural stimulation nad 1.5 hrs binaural stimulation by rhythmical noise signals. The volume of functioning neurons of the nuclei studied was observed either to increase or not to differ from the control. The increase in RNA quantity both in ipsi-, and contralateral medial and lateral nuclei permits making the conclusion on the uniform distribution of binaurally converging connections on the neurons of these nuclei. Moreover, the data obtained enable us to assume that the accumulation of cytoplasmatic RNA occurs not only during excitation but also during inhibition.

Animals↗
PubMed1978

Chemical and conformational changes in chromosome regions being actively transcribed.

U.V. microspectrophotometry has been used to calculate quantities of nucleic acids and proteins of complete polytene chromosomal sets and specific regions of these chromosomes. It has been found that in chromosomes the ratio of DNA to proteins is approximately 1:4. This ratio however changes when specific regions are compared. The average ratio of DNA to proteins in a puffed region (2-48B4C5) increases to 1:16 in contrast to 1:6 from the same region but in non puffed state. At the same time the RNA quantity increases by a factor of 2. thermal denaturation profiles of formaldehyde fixed chromosomes show that the Tm of this region in puffed and non puffed state differ by 10 degrees C. Moreover these profiles suggest that a large fraction of histone-bound DNA is destabilized during puffing.

Animals↗
PubMed1994

[Titration and genotyping of hepatitis C virus RNA in chronic hepatitis C patients treated with interferon].

In order to evaluate the relation of the response to IFN therapy with viral factors, we quantified and typed HCV-RNA in sera and liver specimens from hepatitis C patients treated with IFN by using the single tube polymerase chain reaction. Sustained serum aminotransferase (sALT) normalization was resulted in 11 (19.6%) of the 51 patients. At 6 months after IFN therapy, serum HCV-RNA was negative in 13 of 44 patients. The pretreatment serum HCV-RNA titers tended to be higher in no responders than in complete responders and partial responders. HCV-RNA concentration in the liver correlated with serum HCV-RNA quantity. HCV infections with type I, II, III, IV, II + III, III + IV or II + III + IV were detected in 0, 24, 9, 2, 9, 2 or 3 patients, respectively. Mixed infection with more than 2 genotypes of HCV was found commonly than previously reported. IFN therapy induced complete response in 6 (66.7%) of the 9 patients with type III infection, and this response rate was apparently higher than that in the patients infected with type II or mixed type of HCV. Serum HCV-RNA titers were significantly higher in the patients with type III infection than in the patients with type II or mixed type infection. These findings implied that the determinations of pretreatment serum HCV-RNA titers and HCV-genotypes could be useful to predict sALT improvement and HCV eradication by IFN therapy.

Adult↗
PubMed1991

Ponderable indices in the offspring and nucleic acids quantity in endocrine glands of rats exposed to real and sham VDU screens.

Adult female and male Imp: DAK rats were exposed beneath a real VDU screen (Erl groups) loaded with light-green characteristics due to computation of trigonometric functions in a loop program from a microcomputer. The control animals were exposed beneath a sham screen (Esh groups), being the front glass of vacuum devoid cathode ray tube covered with a transparent celluloid sheet printed with light-green characteristics. The difference in body weight of foetuses from autopsied Erl and Esh females was due mainly to a one-day delay in the fecundation of Esh females. In other groups of Erl females, a prolongation of the oestrous cycle was found in comparison to that of the Esh females. In both Erl females and Erl males, a nonsignificant decrease of weight of anterior pituitary and adrenals was found. RNA quantity was augmented in the anterior pituitary of Erl females and DNA quantity was diminished in the hypothalamus of Erl males. The obtained results suggest the suppression of hypothalamo-hypophyseal function in rats exposed to a real VDU screen.

Animals↗
PubMed2006

Preanalytical aspects of quantitative TaqMan real-time RT-PCR: applications for TF and VEGF mRNA quantification.

OBJECTIVES: The present paper focuses on preanalytical aspects of tissue factor (TF) and vascular endothelial growth factor (VEGF) mRNA quantification: the choice of blood collection tubes and defining the time frame allowed before processing the sample. DESIGN AND METHODS: Blood was collected from healthy volunteers in K(3) EDTA tubes, CPT, endotoxin-free EndoTube tubes and in PAXgene tubes. Total RNA concentration was determined by absorbance readings at 260 nm with a GeneQuantII UV spectrophotometer. RNA quantity and quality were also determined by the Lab on a Chip technique (Agilent 2100 Bioanalyzer). Real-time RT-PCR assays were performed by the TaqMan technology. RESULTS: The more expensive PAXgene and CPT tubes and the Endo tubes did not give superior results from those obtained in inexpensive routine K(3) EDTA tubes. The PAXgene tubes preserved high molecular mass rRNA better than the other tubes. CONCLUSION: Both the PAXgene system and routine EDTA tubes are suitable for clinical purposes aimed at quantitation of mRNA for TF and VEGF. PAXgene yielded rRNA that was less degraded but had lower mRNA per microg extracted RNA. A time frame up to 24 h until sample processing is acceptable for TF and VEGF mRNA.

DNA, Complementary↗
PubMed2002

Variation of hepatitis C virus load, hypervariable region 1 quasispecies and CD81 hepatocyte expression in hepatocellular carcinoma and adjacent non-cancerous liver.

Hepatitis C virus (HCV) infection is etiologically associated with the development of hepatocellular carcinoma (HCC) worldwide. HCV has been reported to exist and replicate in both HCC and adjacent non-cancerous liver tissue, but limited information was available on HCV viral load and quasispecies composition in HCC relative to adjacent non-cancerous hepatocytes. Previous study has also suggested CD81, a surface hepatocyte protein, as a receptor for HCV. To clarify the above, HCV-RNA and CD81-RNA titers in 20 paired hepatectomized liver and serum were quantitatively measured by chemiluminescent RT-cPCR. Hypervariable region 1 (HVR-1) variations of parallel specimens were analyzed after subcloning in 6 patients. HCV-RNA levels in serum and non-cancerous liver were markedly higher for HCV genotype 1 than genotype non-1. HCV levels were markedly higher in non-cancerous liver than in HCC (P = 0.001) in a genotype-independent manner, with a mean ratio of 56:1 for non-cancerous tissue to HCC. Both non-cancerous and HCC tissues had the same level of CD81-RNA expression, which was not linked to HCV load. HCV-RNA quantity in both HCC and non-cancerous liver correlated with the number of HVR-1 quasispecies in the tissue, and distinct HVR-1 subclones existed.

Adult↗
PubMed2005

Outcome analysis of patients with squamous cell carcinoma of the head and neck and hepatitis C virus.

OBJECTIVE/HYPOTHESIS: Infection with the hepatitis C virus (HCV) is a global problem with over 170 million people infected. Recently, we have noticed that a large number of patients diagnosed with squamous cell carcinoma of the head and neck (SCCHN) have also been diagnosed with HCV. A review of the literature reveals little information concerning this patient population. The objective of this study was to compare the outcome of SCCHN patients who have been exposed to HCV with naïve SCCHN patients. STUDY DESIGN: Retrospective chart review. METHODS: A retrospective chart review from June 1991 through December 2002 was performed to identify patients diagnosed with SCCHN who were screened for HCV. Patients were stratified into two groups (HCV positive and HCV negative). Data were recorded on patients for status of disease at last clinic visit, pretreatment serum albumin and hematocrit levels, and RNA quantities of HCV. Statistical analysis was performed using paired t test to compare serum albumin and hematocrit levels. Kaplan-Meier survival curves were used to compare outcomes. The log-rank test was used to determine significance. Cox regression was used to examine the association of prognostic predictor variables with overall survival and disease-free survival. RESULTS: There was no difference noted in 5 year survival between hepatitis C positive and hepatitis C negative groups in overall outcomes (66.7% vs. 67.9%, P = 1.000) or 5 year disease-free survival (90.5% vs. 80.8%, P = .514). The two groups, HCV positive versus HCV negative, also had similar serum albumin levels (3.62 g/dL vs. 3.72 g/dL, P = .37) as well as serum hematocrit levels (42.9% vs. 41.0%, P = .12). Serum levels of hepatitis C RNA were obtained in seven patients, with only one being undetectable. The only prognostic predictor variable that was significantly associated with overall survival was age. None of the predictor variables were significantly associated with disease-free survival. CONCLUSION: Co-infection with HCV, although prevalent in the Veterans Administration Hospital population, did not affect patient outcome as defined by disease-free survival. Patients who were seropositive for HCV had comparable serum albumin levels as well as serum hematocrit when compared with HCV negative patients.

Aged↗
PubMed1997

High levels of serum interleukin-10 are associated with a poor response to interferon treatment in patients with chronic hepatitis C.

BACKGROUND: It still remains unclear whether some immunologic factors affect the response to interferon treatment. We therefore examined whether the pretreatment levels of serum interleukin-10 and soluble intercellular adhesion molecule-1 can be associated with the response to interferon treatment in patients with chronic hepatitis C. METHODS: One hundred and two patients with chronic hepatitis C treated with interferon alpha-2b were divided into three groups on the basis of patterns of biochemical interferon response. Pretreatment levels of serum interleukin-10 and soluble intercellular adhesion molecule-1 were determined using enzyme-linked immunosorbent assay. Hepatitis C virus (HCV) typing was performed with a serologic enzyme-linked immunosorbent assay. RESULTS: For patients with serotype I (n = 76) the numbers of sustained, transient, and non-responders were 12 (16%), 43 (56%), and 21 (28%), respectively. In serotype-I patients the pretreatment levels of serum interleukin-10 in non-responders were significantly higher than those in sustained or transient responders, although no significant differences were observed in HCV RNA quantity between them. There were no significant differences in the pretreatment levels of serum soluble intercellular adhesion molecule-1 among the three groups. CONCLUSION: These findings suggest that high serum interleukin-10 levels may be related to a poor response to interferon treatment in serotype-I patients with chronic hepatitis C.

Adult↗
PubMed1996

Expression of the messenger ribonucleic acids for insulin-like growth factor-I and insulin-like growth factor binding proteins in porcine corpora lutea.

In the pig, the corpus luteum (CL) can develop and function autonomous of pituitary gonadotropins for approximately 12 days. We hypothesized that the insulin-like growth factor (IGF) system may play an autocrine/paracrine luteotrophic role(s) during this period. In this study, we monitored the expression (i.e., steady-state levels of mRNAs) of IGF-I and IGF binding proteins (IGFBP)-2, -3, -4, -5, and -6 mRNAs in whole CL and in small and large luteal cells on Days 4-16 of the estrous cycle. CL were dissociated with collagenase, and large and small luteal cells were isolated by centrifugal elutriation. Whole CL and luteal cells were extracted to isolate total or poly(A)+ RNA, which was subjected to Northern and/or dot-blot analyses using [32P]-labeled cDNA probes for IGF-I and IGFBP-2, -3, -4, -5, and -6. Northern blots showed readily detectable transcripts for IGF-I (6.7 and 0.9 kb), IGFBP-2 (1.8 kb), IGFBP-3 (2.8 kb), IGFBP-4 (2.6 kb), and IGFBP-5 (6.0 kb), but not for IGFBP-6. IGFBP-3 and -5 transcripts were observed mainly in small luteal cells, while IGFBP-2 and -4 were seen in both cell types. Dot-blot analyses for IGF-I and IGFBP-3 mRNAs were performed on total RNA from small and large luteal cells; blots were counter-probed with 3-phosphoglyceraldehyde dehydrogenase (p-GAD) cDNA to assess RNA quantity and quality. IGF-I mRNA (ratio IGF-I:p-GAD mRNA) expression was approximately 2-fold greater in small than in large luteal cells on Days 4-10. However, steady-state levels of IGF-I mRNA in small, but not large, luteal cells decreased significantly on Days 12-16 (vs. Days 4-10). IGFBP-3 mRNA expression was significantly greater (approximately 3-fold) in small than in large luteal cells but did not vary significantly between Days 4-10 and 12-16 for either cell type. We conclude that porcine CL express mRNAs for IGF-I and IGFBP-2, -3, -4, and -5, and that while small luteal cells are the major sources of IGF-I and IGFBP-3 and -5, IGFBP-2 and -4 appear to be expressed to approximately the same extent in small and large luteal cells. These results further suggest that the IGF-I/IGF system may have autocrine/paracrine regulatory actions in CL development/function in the pig.

Animals↗
PubMed2002

Molecular staging of lung cancer: real-time polymerase chain reaction estimation of lymph node micrometastatic tumor cell burden in stage I non-small cell lung cancer--preliminary results of Cancer and Leukemia Group B Trial 9761.

OBJECTIVE: The 5-year survival for patients with surgically resected stage I non-small cell lung cancer is only 60% to 70%, probably because of undetected systemic occult micrometastases. Detection of occult micrometastases in lymph nodes by reverse-transcriptase polymerase chain reaction for carcinoembryonic antigen messenger RNA in non-small cell lung cancer has not been reported. Detection of occult micrometastases by standard reverse-transcriptase polymerase chain reaction provides only yes or no answers about their presence, whereas quantitative real-time reverse-transcriptase polymerase chain reaction permits reproducible quantitation of target molecules. This study evaluated the ability of quantitative reverse-transcriptase polymerase chain reaction to quantitate lymph node occult metastases with carcinoembryonic antigen messenger RNA as a tumor marker. METHODS: Standard reverse-transcriptase polymerase chain reaction and quantitative reverse-transcriptase polymerase chain reaction for carcinoembryonic antigen messenger RNA were performed on 232 lymph nodes from 53 patients with stage I disease (node negative according to histologic examination). Quantitative reverse-transcriptase polymerase chain reaction determined carcinoembryonic antigen messenger RNA quantity by detecting fluorescence increase at a threshold polymerase chain reaction cycle. Threshold polymerase chain reaction cycle values were correlated with standard curves created from serially diluted carcinoembryonic antigen-positive HTB-174 tumor cells to estimate the number of micrometastatic tumor cells in a lymph node. RESULTS: Detection rates of occult metastases were similar for standard reverse-transcriptase polymerase chain reaction and quantitative reverse-transcriptase polymerase chain reaction at 38 of 232 (16.4 %) and 59 of 232 (25.4 %), respectively. Upstaging rates among 53 cases of stage I non-small cell lung cancer were also similar for standard reverse-transcriptase polymerase chain reaction and quantitative reverse-transcriptase polymerase chain reaction at 23 of 53 (43.4 %) and 30 of 53 (56.6%), respectively. Comparison of positive lymph node stations according to quantitative reverse-transcriptase polymerase chain reaction (threshold polymerase chain reaction cycle <45) with HTB-174 tumor cell standard curves yielded estimates of metastatic tumor cell burden of 1.07 x 10(3)to 3.24 x 10(5)cells per lymph node station (median 7190 tumor cells per lymph node station). CONCLUSIONS: Standard and quantitative real-time reverse-transcriptase polymerase chain reaction for carcinoembryonic antigen detected occult metastases in patients with stage I non-small cell lung cancer at similar rates; both upstaged about 50% of cases. Quantitative reverse-transcriptase polymerase chain reaction allows estimation of the number of metastatic cells per lymph node, however, which potentially allows greater precision in predicting recurrence risk.

Adenocarcinoma↗
PubMed2003

Tumor suppressor role of KiSS-1 in bladder cancer: loss of KiSS-1 expression is associated with bladder cancer progression and clinical outcome.

The expression profiles of nine bladder cancer cell lines were compared against a pool containing equal total RNA quantities of each of them. Lower expression of KiSS-1 was revealed in cells derived from the most advanced bladder tumors. When comparing 15 primary bladder tumors versus a pool of four bladder cancer cell lines, lower transcript levels of KiSS-1 were observed in the invasive bladder carcinomas as compared to superficial tumors. KiSS-1 expression ratios provided prognostic information. The expression pattern of KiSS-1 transcripts was analyzed using in situ hybridization in nine bladder cancer cells, paired normal urothelium and bladder tumor samples (n = 25), and tissue microarrays of bladder tumors (n = 173). We observed complete loss of KiSS-1 in all invasive tumors under study as compared to their respective normal urothelium. The expression of KiSS-1 was found to be significantly associated with histopathological stage. Patients with lower KiSS-1 expression showed a direct correlation with overall survival in a subset of bladder tumors whose follow-up was available (n = 69). We did not observe any significant differential KiSS-1 expression along cell cycle by sorting analysis. A potential tumor suppressor role in bladder cancer was revealed for KiSS-1. Moreover, it showed predictive value by identifying patients with poor outcome.

Adult↗
PubMed2004

Natural interferon alpha treatment and interferon alpha receptor 2 levels in acute hepatitis C.

Efficacy of interferon (IFN) therapy during the acute phase of hepatitis C infection is promising, although the optimal regimen has yet to be determined. It is not known whether the known prognostic factors for chronic hepatitis C (CHC) influence the effect of IFN in acute hepatitis C (AHC). Seventeen patients with AHC were analyzed for hepatic IFN alpha receptor 2 (IFNAR2) prior to IFN treatment. All patients were subsequently treated with either 168 million units (MU) or 336 MU of natural IFN alpha. Seventeen age-matched samples of CHC were provided as controls. The overall sustained response rate was 64.7% (11/17). In patients who received a total dose of 168 MU IFN, the sustained response rate was 28.6% (2/7), and in those who received 336 MU of IFN, the sustained response rate was 90.0% (9/10). The peaks of ALT and HCV-RNA quantity were not associated with the response to IFN. The hepatic IFNAR2 levels were 1.52 +/- 0.34 densitometry units and 0.92 +/- 0.16 in AHC and CHC, respectively (P = 0.042). There was no difference in hepatic IFNAR2 levels between sustained virological responders (SVR) and nonsustained virological responders (NR). The hepatic receptor levels were higher in AHC than in CHC patients. The levels of hepatic IFNAR2 did not differ in SVR and NR, indicating that high-dose natural IFN alpha treatment is effective for AHC, irrespective of the levels of hepatic IFNAR2.

Acute Disease↗