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At least 181 records · Page 10Linked to original sources
PubMed2001

Identification of differentially expressed genes in organ-confined prostate cancer by gene expression array.

BACKGROUND: To understand the molecular mechanisms underlying prostate cancer, we have utilized the gene expression array to search for genes whose expression is altered in this disease. METHODS: RNA quality from manual microdissected tissue was compared with that from microselected tissue by electrophoresis. For array analysis, malignant and normal prostate epithelium was enriched using microselection technique from prostate cancer and the peripheral zone of a normal prostate. Identical array membrane was hybridized to labeled cancer and normal cDNA, respectively. The differentially expressed gene was further evaluated by RT-PCR. RESULTS: Microdissection, but not microselection, causes visible degradation to RNA. Of the 588 genes on the membrane, 87 genes yielded significant signals. Based on a three fold difference relative to normal prostate tissue, 1 gene was overexpressed and 12 genes underexpressed in prostate cancer. Of them, five showed statistically significant reduction in mRNA levels in six prostate cancer specimens compared with seven normal prostate specimens. These five genes are glutathione S-transferase M1 (GSTM1), monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor-alpha receptor-1 (TNFR-1), transforming growth factor beta3 (TGF-beta3), and inhibitor of DNA binding-1 (ID-1). CONCLUSIONS: GST-based metabolism, cytokine MCP-1 and TNFR-1, and TGF-beta3 signaling pathways, and some helix-loop-helix nuclear proteins could be potentially important in organ-confined prostate cancer and deserve further investigation.

Adult↗
PubMed2003

A simple and efficient Triton X-100 boiling and chloroform extraction method of RNA isolation from Gram-positive and Gram-negative bacteria.

A fast, reliable, and inexpensive Triton X-100 boiling procedure for RNA isolation from both the Gram-positive and Gram-negative bacteria was developed. The yield of RNA was 0.2-2 mg per 10 ml bacterial culture. The method was tested on Gram-positive and Gram-negative bacteria of eight genera and nine species and yielded reproducible results. In parallel experiments, the Qiagen and hot phenol extraction methods both yielded RNA that contained contaminating 16S and 23S rRNA. The Triton X-100 boiling method reported here yielded RNA that was free from 16S and 23S rRNA, contained full-length transcripts and did not require additional purification. The presence of specific mRNA in one of the RNA samples obtained by this procedure was demonstrated by partial amplification of a 732 bp vancomycin resistance gene, vanA, by reverse transcription-polymerase chain reaction (RT-PCR). The presence of a full-length transcript (1031 bases) of the vanA gene was verified by Northern hybridization and probing with a digoxigenin (DIG)-labeled vanA PCR partial product. The method provides a rapid, reliable, and simple tool for the isolation of good quality RNA suitable for various molecular biology experiments.

Chloroform↗
PubMed2006

RCL2, a new fixative, preserves morphology and nucleic acid integrity in paraffin-embedded breast carcinoma and microdissected breast tumor cells.

Methacarn and RCL2, a new noncrosslinking fixative, were compared to formalin-fixed or frozen tissue samples of the same invasive breast carcinoma and were evaluated for their effects on tissue morphology and immunohistochemistry as well as DNA and RNA integrity. The histomorphology of methacarn- or RCL2-fixed paraffin-embedded tumors was similar to that observed with the matched formalin-fixed tissues. Immunohistochemistry using various antibodies showed comparable results with either fixative, leading to accurate breast tumor diagnosis and determination of estrogen and progesterone receptors, and HER2 status. Methacarn and RCL2 fixation preserved DNA integrity as demonstrated by successful amplification and sequencing of large DNA amplicons. Similarly, high-quality RNA could be extracted from methacarn- or RCL2-fixed paraffin-embedded MCF-7 cells, whole breast tumor tissues, or microdissected breast tumor cells, as assessed by electropherogram profiles and real-time reverse transcriptase-polymerase chain reaction quantification of various genes. Moreover, tissue morphology and RNA integrity were preserved after 8 months of storage. Altogether, these results indicate that methacarn, as previously shown, and RCL2, a promising new fixative, have great potential for performing both morphological and molecular analyses on the same fixed tissue sample, even after laser-capture microdissection, and can open new doors for investigating small target lesions such as premalignant breast lesions.

Breast Neoplasms↗
PubMed2006

Expression profiling of intermingled long-range projection neurons harvested by laser capture microdissection.

Gene expression data are most useful if they can be associated with specific cell types. This is particularly so in an organ such as the brain, where many different cell types lie in close proximity to each other. We used zebra finches (Taeniopygia guttata), fluorescent tracers and laser capture microdissection (LCM) to collect projection neurons and their RNAs from two interspersed populations from the same animal. RNA amplified from each cell class was reverse transcribed, fluorescently labeled, and hybridized to cDNA microarrays of genes expressed in the zebra finch brain. We applied strict fold-expression criteria, supplemented by statistical analysis, to single out genes that showed the most extreme and consistent differential expression between the two cell classes. Confirmation of the true expression pattern of these genes was made by in situ hybridization and Taqman quantitative PCR (qPCR). High quality RNA was obtained, too, from backfilled neurons birth-dated with bromodeoxyuridine (BrdU). We also quantified changes in the levels of three genes after singing behavior using qPCR. Thus, we have brought together a combination of techniques allowing for the molecular profiling of intermingled populations of projection neurons of known connectivity, age and experience, which should constitute a powerful tool for CNS research.

Animals↗
PubMed2006

Rhodamine-RCA in vivo labeling guided laser capture microdissection of cancer functional angiogenic vessels in a murine squamous cell carcinoma mouse model.

BACKGROUND: Cancer growth, invasion and metastasis are highly related to tumor-associated neovasculature. The presence and progression of endothelial cells in cancer is chaotic, unorganized, and angiogenic vessels are less functional. Therefore, not all markers appearing on the chaotic endothelial cells are accessible if a drug is given through the vascular route. Identifying endothelial cell markers from functional cancer angiogenic vessels will indicate the accessibility and potential efficacy of vascular targeted therapies. RESULTS: In order to quickly and effectively identify endothelial cell markers on the functional and accessible tumor vessels, we developed a novel technique by which tumor angiogenic vessels are labeled in vivo followed by Laser Capture Microdissection of microscopically isolated endothelial cells for genomic screening. Female C3H mice (N = 5) with established SCCVII tumors were treated with Rhodamine-RCA lectin by tail vein injection, and after fluorescence microscopy showed a successful vasculature staining, LCM was then performed on frozen section tissue using the PixCell II instrument with CapSure HS caps under the Rhodamine filter. By this approach, the fluorescent angiogenic endothelial cells were successfully picked up. As a result, the total RNA concentration increased from an average of 33.4 ng/ul +/- 24.3 (mean +/- S.D.) to 1913.4 ng/ul +/- 164. Relatively pure RNA was retrieved from both endothelial and epithelial cells as indicated by the 260/280 ratios (range 2.22-2.47). RT-PCR and gene electrophoresis successfully detected CD31 and Beta-Actin molecules with minimal Keratin 19 expression, which served as the negative control. CONCLUSION: Our present study demonstrates that in vivo Rhodamine RCA angiogenic vessel labeling provided a practical approach to effectively guide functional endothelial cell isolation by laser capture microdissection with fluorescent microscopy, resulting in high quality RNA and pure samples of endothelial cells pooled for detecting genomic expression.

Animals↗
PubMed1995

Prognostic significance of the RT-PCR assay of PML-RARA transcripts in acute promyelocytic leukemia. The Leukemia Study Group of the Ministry of Health and Welfare (Kouseisho).

Minimal residual disease (MRD) was prospectively monitored at the 10(-5) level by the reverse transcriptase-polymerase chain reaction (RT-PCR) of PML-retinoic acid receptor alpha (RARA) transcripts from 27 acute promyelocytic leukemia (APL) patients who achieved complete remission (CR) with all-trans retinoic acid and chemotherapy (previously untreated patients, 15; refractory to chemotherapy or relapsed, 12). The RNA quality from bone marrow cells was firstly assessed by gel electrophoresis to avoid false negativity because of the fragility of the APL cells and the PML-RARA transcripts. In 12 of 15 untreated patients, RT-PCR became negative during consolidation and intensification therapy 4-16 months after the initiation of therapy, whereas it remained positive in nine of 12 refractory patients. At the end of therapy, RT-PCR was negative in 14 patients and positive in 13 patients. The former patients remained in CR at median follow-up of 9 months after the end of therapy. In the latter, however, 10 patients relapsed at a median of 5 months after the end of therapy. These results suggest that the RT-PCR assay can evaluate the quality of CR in APL and predict subsequent relapse.

Adult↗
PubMed2004

cDNA microarray analysis with amplified RNA after isolation of intact cellular RNA from neoplastic and non-neoplastic prostate tissue separated by laser microdissections.

Laser microdissection is a valuable tool to prepare pure cell populations from complex tissues for further analyses. Gene expression studies by real-time RT-PCR and cDNA arrays of microdissected tissues are becoming widely used methods. The integrity and quantity of prepared RNA must be proven to ensure reliable results in subsequent applications such as quantitative RT-PCR and cDNA-arrays. In the present study we used RNAlater trade mark protected prostate tissue for laser microdissection of tumor and tumor-free tissues. RNA quality and quantity was assessed using automated capillary gel electrophoresis. By using quantitative real time-RT-PCR before and after mRNA amplification the insulin-like growth factor binding protein-3 (IGFBP-3) gene expression was shown to be down-regulated in three out of five cases and DD3 was up-regulated in cancer tissues in all cases. The up-regulation of DD3 and the down-regulation of IGFBP-3 gene expression in cancer tissue were conserved after RNA amplification. A cDNA microarray also revealed an IGFBP-3 down-regulation in cancer tissue as well as several genes known to be differerentially expressed in prostate cancer. Taken together, we present a novel method for the isolation of intact RNA from laser microdissection-derived prostate cancer tissue useful for downstream applications of real-time RT-PCR and cDNA microarrays.

Antigens, Neoplasm↗
PubMed2001

RNA recovery and detection of mRNA by RT-PCR from preserved prokaryotic samples.

The effectiveness of maintaining prokaryotic RNA in Synechococcus and Pseudomonas cells, fixed in 96% ethanol, 4% paraformaldehyde, or suspended in RNAlater, and held in cold storage for 3 months was compared. Fluorometric determination of the RNA extracted from Synechococcus and Pseudomonas cells indicated that the cell storage treatments tested were equally effective at maintaining their total RNA content. There was not any detectable decrease in the quantity of RNA isolated from the preserved samples during storage. Intact mRNA transcripts of the RuBisCO (rbcL) and nir genes were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) from preserved bacterial cells throughout 3 months of storage. In contrast, RT-PCR failed to amplify the mRNA of the rbcL and nitrite reductase genes in unfixed and/or unpreserved bacterial samples, suggesting that bacterial mRNA can be well maintained during a prolonged storage when cells are preserved properly. In addition, RNAlater is a useful reagent for the storage and maintenance of high quality RNA in unfrozen samples.

Bacteria↗
PubMed2004

Single muscle fiber gene expression in human skeletal muscle: validation of internal control with exercise.

Reverse transcription and real-time PCR have become the method of choice for the detection of low-abundance mRNA transcripts obtained from small human muscle biopsy samples. GAPDH, beta-actin, beta-2M, and 18S rRNA are widely employed as endogenous control genes, with the assumption that their expression is unregulated and constant for given experimental conditions. The aim of this study was to determine if mRNA transcripts could be performed on isolated human single muscle fibers and to determine reliable housekeeping genes (HKGs) using quantitative gene expression protocols at rest and in response to an acute exercise bout. Muscle biopsies were obtained from the gastrocnemius of three adult males before, immediately after, and 4 h following 30 min of treadmill running at 70% of VO2max. A total of 40 single fibers (MHC I and IIa) were examined for GAPDH, beta-actin, beta-2M, and 18S rRNA using quantitative RT-PCR and SYBR Green detection. All analyzed single fiber segments showed ribosomal RNA (28S/18S). No degradation or additional bands below ribosomal were detected (rRNA ratio 1.5-1.8). Also, no high or low-molecular weight genomic DNA contamination was observed. For each housekeeping gene the duplicate average SD was +/-0.13 with a CV of 0.58%. Stable expression of GAPDH was observed at all time points for each fiber type (MHC I and IIa). Inconsistent expression of beta-actin, beta-2M, and 18S rRNA was observed during the post-exercise time points for each fiber type. These data indicate that successful extraction of high quality RNA from human single muscle fibers along with quantification of mRNA of selected genes can be performed. Furthermore, exercise does influence the expression of certain HKGs with GAPDH being the most stable.

Adaptation, Physiological↗
PubMed2003

Quantification of mRNA degradation as possible indicator of postmortem interval--a pilot study.

Postmortem RNA degradation is a complex process which has not been studied systematically up to now. RNA, in contrast to DNA, is degraded in vivo to control translation and is believed to be less stable than DNA in vitro and postmortem due to ubiquitously present ribonucleases. Quantification of the fragmentation level of human mRNA might be a tool for determination for the time since death. We have developed a method for quantitating mRNA degradation by multiplex-RT-PCR coupled to laser-induced fluorescence capillary electrophoresis. The study included whole blood samples from living individuals and postmortem blood and brain samples which were stored refrigerated for up to 5 days. It could be shown that RNA degradation is significantly correlated with the storage interval of blood samples taken from living individuals and with the postmortem interval (PMI) in autopsy cases. This technique might become a parameter for the assessment of RNA quality in postmortem samples used for diagnosis and research and for the determination of the PMI. However, rather large confidence intervals have to be considered so that the use of this method in forensic casework requires further research.

Adolescent↗
PubMed1997

Methods to prepare RNA and to isolate developmentally regulated genes from Eimeria.

Coccidians represent a large class of important intracellular parasites that traverse multiple developmental stages that are distinct and required to complete the life cycle. The biochemical details underlying the regulation of transformation from one developmental form to the next are limited and the study of such details presents unique obstacles. However, the genetic program is critical and may provide a basis for understanding the biology of these organisms in addition to the opportunity to suppress development and infection. We provide a basic overview of several strategies, including previously unpublished results, used by this laboratory to isolate stage-specific genes from Eimeria bovis. Additionally, we have included detailed discussions that summarize the associated advantages and disadvantages of each as applied to coccidia and potentially to other parasites in the phylum Apicomplexa. Given that the purification of sufficient quantities of high-quality RNA is vital, we have included detailed protocols for the isolation of RNA from various parasite stages. Also included is a detailed protocol to apply mRNA differential display to investigate stage-specific developmental regulation.

Animals↗
PubMed2005

RNA integrity and in situ RT-PCR in dento-alveolar tissues after microwave accelerated demineralisation.

OBJECTIVE: The structural organization of oral soft tissue and its relationship with highly calcified teeth are difficult to preserve unless tissues are decalcified, paraffin embedded and subsequently sectioned. However, enamel decalcification time and its negative impact on RNA integrity makes it difficult to effectively analyse in situ gene expression. This study examined the impact of microwave-enhanced decalcification on processing time, RNA integrity and detection of in situ mRNA expression in hard and soft tissue for cell type specific markers of Keratinocyte growth factor receptor, Scleraxis and Osteonectin. DESIGN: Maxillas and mandibles were obtained from three male Wistar strain rats. Right side tissues were decalcified using a microwave plus 10% EDTA solution (M+) while left side tissues were decalcified in 10% EDTA solution alone (M-). RESULTS: Microwave use reduced decalcification time by up to 50% and had no significant impact on morphology, RNA quality and in situ detection of gene expression relative to the M-group. CONCLUSIONS: In situ RT-PCR gene expression of microwave decalcified paraffin-embedded oral tissues is an effective technique to localize in situ gene expression while maintaining excellent soft and hard tissue architecture.

Animals↗
PubMed2002

A rapid and reliable enzyme immunoassay PCR-based screening method to identify EBV-carrying gastric carcinomas.

Epstein-Barr virus (EBV) is associated with a substantial number of gastric adenocarcinomas worldwide, as confirmed by EBER1/2-RNA in situ hybridization (RISH). In the present study, we developed a rapid and sensitive PCR-based prescreening method for the detection of EBV in gastric carcinomas to reduce the amount of laborious EBER1/2-RISH assays to be performed. The method was evaluated by testing gastric adenocarcinomas (n = 242) using both BamHI W PCR-enzyme immunoassay (EIA) and EBER1/2-RISH, in combination with appropriate DNA and RNA quality controls. Seventy-four percent of the paraffin-embedded gastric adenocarcinomas had good DNA quality as shown by beta-globin polymerase chain reaction (PCR) after proteinase K and boiling pretreatment, whereas after DNA purification this was increased to 90%. Thirty-two percent of all cases were EBV-DNA positive after PCR-EIA, whereas 10% of these gastric cancers contained EBV transcripts in the neoplastic cells as confirmed by EBER1/2-RISH. Interestingly, only samples with high optical density (OD) 405/630 values in PCR-EIA, equivalent to the maximum reading of the assay as determined by the positive control, contained EBV-positive tumor cells in the EBER1/2-RISH. In contrast, the weak positive samples, as determined by low OD readings in the PCR-EIA were EBER1/2-RISH negative. In conclusion, high OD values in EBV PCR-EIA are very valuable to prescreen EBV-carrying gastric carcinomas as confirmed by EBER1/2-RISH. Only these samples and those with poor DNA quality will require testing in the EBER1/2-RISH, thereby reducing the amount of laborious RISH assays with 85%.

Actins↗
PubMed1990

Detection and assessment of severity of tricuspid regurgitation using first-pass radionuclide angiography and comparison with pulsed Doppler echocardiography.

Noninvasive detection and semiquantitative assessment of tricuspid regurgitation (TR) were performed using first-pass radionuclide angiography (RNA), by sampling a region of interest over the right atrium for any tracer entering the right atrium during right ventricular systole. The relative amount of tracer entering the right atrium was quantitated and the results were compared with semiquantitative Doppler echocardiographic grading of TR severity. Using the right ventricular time-activity curve to define end-diastolic and end-systolic frames, the right atrial counts for the 2 or 3 cardiac cycles after the peak right ventricular counts were summed. The right atrial "injection fraction" was calculated using the following formula: [(end-systolic counts - end-diastolic counts)/(end-diastolic counts)] X 100%. The right atrial injection fraction was examined in 51 patients who had good quality RNA and Doppler studies. Of 27 patients with no evidence of TR by Doppler, 26 had a negative right atrial injection fraction. All 24 patients with a positive Doppler for TR had a positive right atrial injection fraction. Comparison of right atrial injection fraction grade ranges with semiquantitative grades of TR severity on Doppler revealed identical grades in 21 of the 24, with a single grade difference in the remaining 3 patients. Thus, right atrial time-activity curve quantitation during routine first-pass RNA allows detection and grading of the severity of TR, with results very similar to pulsed Doppler echocardiography. This simple procedure is easily appended to the evaluation of ventricular performance with first-pass RNA.

Adolescent↗
PubMed1997

Multiprimed cDNA synthesis followed by PCR is the most suitable method for Epstein-Barr virus transcript analysis in small lymphoma biopsies.

In this study, the reverse transcriptase-polymerase chain reaction (RT-PCR) for the reliable detection of multiple Epstein-Barr virus (EBV) transcripts was optimized and subsequently evaluated on lymphoma specimens. Since often only small lymphoma biopsies are available for analysis of EBV transcripts, several RT-protocols to generate cDNA from multiple targets were applied. These were multi-primer, oligo-dT primed and random hexamer primed cDNA synthesis. Multi-primer cDNA synthesis appeared to be the most suitable method for subsequent PCR analysis of EBV targets; simultaneous priming with up to 10 specific antisense primers (for EBNA1 and 2, LMP1 and 2, BARF0, BHRF1, BZLF1, C promoter activity and the RNA control genes U1A and c-abl) followed by PCR showed no loss of sensitivity compared to single-specific antisense priming. Transcripts were specifically detected in up to one EBV-positive JY cell in a background of 50,000 EBV-negative BJAB cells, with the exception of BZLF1 and QK spliced EBNA1 transcripts which could only be detected in 1000 and 10,000 EBV-positive cells, respectively. The analytical sensitivities of all the primers used in PCR, including BZLF1 and QK EBNA1 primers, were 1-10 copies of cloned RT-PCR products. The multi-primed RT-PCR was evaluated on lymphomas (n = 13). In cases with proper RNA quality, EBV expression patterns found were identical to those found in previous studies using single-primed RT-PCR assays. In conclusion, this study shows that multi-primed RT-PCR analysis can be used efficiently for EBV transcript analysis in small lymphoma biopsies, thereby facilitating studies concerning the role of EBV in lymphomagenesis.

Antisense Elements (Genetics)↗
PubMed2004

The Ro 60 kDa autoantigen: insights into cellular function and role in autoimmunity.

An RNA-binding protein, the Ro 60 kDa autoantigen, is a major target of the immune response in patients suffering from two systemic rheumatic diseases, systemic lupus erythematosus and Sjogren's syndrome. In lupus patients, anti-Ro antibodies are associated with photosensitive skin lesions and with neonatal lupus, a syndrome in which mothers with anti-Ro antibodies give birth to children with photosensitive skin lesions and a cardiac conduction defect, third degree heart block. In vertebrate cells, the Ro protein binds small RNAs of unknown function known as Y RNAs. Although the cellular function of Ro has long been mysterious, recent studies have implicated Ro in two distinct processes: small RNA quality control and the enhancement of cell survival following exposure to ultraviolet irradiation. Most interestingly, mice lacking the Ro protein develop an autoimmune syndrome that shares some features with systemic lupus erythematosus in patients, suggesting that the normal function of Ro may be important for the prevention of this autoimmune disease. In this review, we summarize recent progress towards understanding the role of the Ro 60 kDa protein and discuss whether the cellular function of Ro could be related to certain manifestations of lupus in patients.

Animals↗
PubMed2005

Normalization of gene expression measurements in tumor tissues: comparison of 13 endogenous control genes.

For interpretation of quantitative gene expression measurements in clinical tumor samples, a normalizer is necessary to correct expression data for differences in cellular input, RNA quality, and RT efficiency between samples. In many studies, a single housekeeping gene is used for normalization. However, no unequivocal single reference gene (with proven invariable expression between cells) has been identified yet. As the best alternative, the mean expression of multiple housekeeping genes can be used for normalization. In this study, no attempt was made to determine the gold-standard gene for normalization, but to identify the best single housekeeping gene that could accurately replace the measurement of multiple genes. Expression patterns of 13 frequently used housekeeping genes were determined in 80 normal and tumor samples from colorectal, breast, prostate, skin, and bladder tissues with real-time quantitative RT-PCR. These genes included, large ribosomal protein, beta-actin, cyclophilin A, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerokinase 1, beta-2-microglobin, beta-glucuronidase, hypoxanthine ribosyltransferase (HPRT), TATA-box-binding protein, transferrin receptor, porphobilinogen deaminase, ATP synthase 6, and 18S ribosomal RNA. Principal component analysis was used to analyze these expression patterns, independent of the level of expression. Our approach identified HPRT as the single best reference gene that could be used as an accurate and economic alternative for the measurement of multiple housekeeping genes. We recommend this gene for future studies to standardize gene expression measurements in cancer research and tumor diagnostics until a definite gold standard has been determined.

Adenocarcinoma↗
PubMed2006

Differential-display analysis of gene expression in livers from normal and partially hepatectomized mice.

Partial hepatectomy, resulting in the removal of approximately 70% of the liver, is widely utilized for studies of liver growth in experimental animals. The regenerative response is proportional to the amount of liver removed. Knowing when and where genes are expressed provides a strong clue as to its biological role. The RNA differential-display (DD) technique facilitates monitoring the differential expression of a large number of activated or suppressed genes under various biological conditions. To reveal mechanisms of liver regeneration, we performed a comparative analysis of gene expression during liver regeneration using DD. We sacrificed male Balb/c mice, aged 10 to 12 weeks, at 0, 24, 48, and 72 hours, and 1 and 2 weeks after PHx. The livers were weighed, and the amount of glutamic-oxaloacetate transaminase in serum measured. We extracted the total RNA from frozen liver tissue and confirmed the RNA quality using a lab-chip system. DD analysis was performed essentially as described by Liang and Pardee. Semiquantitative reverse-transcription polymerase chain reaction was performed to confirm the results of DD analysis. Of the 56 fragments that exhibited changed expression levels during PHx, 39 were cloned and sequenced. There were 31 known genes, 13 unknown genes, and 9 expressed-sequence tags. These results indicated that DD is a powerful approach for monitoring molecular events in the regenerating liver.

Animals↗