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At least 307 records · Page 17Linked to original sources
PubMed2006

Extraction of RNA from archival tissues and measurement of thrombospondin-1 mRNA in normal, dysplastic, and malignant oral tissues.

Thrombospondin-1 (TSP-1) is an extracellular matrix glycoprotein implicated in the regulation of angiogenesis and tumour development. Our objectives were to ascertain the quantity and quality of RNA extracted from archival, formalin-fixed, paraffin embedded, oral tissues and their application in measuring the concentrations of TSP-1 mRNA in these tissues. We compared three techniques of isolation of RNA as well as related experimental variables. TSP-1 mRNA was measured in specimens of normal, dysplastic, and malignant oral tissues by real-time reverse transcriptase polymerase chain reaction (RT-PCR). RNA suitable for analysis by real-time RT-PCR was obtained by the three techniques tested, although the yield varied depending on the protocol used (range 0.2-3.6 microg/mm(3)). The mean (S.D.) concentrations of TSP-1 mRNA relative to 18S were 21.1 (7.2) in normal oral tissues (n=9), 11.0 (8.2) in dysplastic tissue (n=8) and 7.3 (5.3) in carcinomatous tissue (n=17). The difference between normal and carcinomatous specimens was significant (p=0.01). This reduction in expression of TSP-1 mRNA from normal to dysplasia to carcinoma may favour the angiogenic drive that accompanies the development of oral tumours.

Carcinoma, Squamous Cell↗
PubMed2002

Primordial genetics: phenotype of the ribocyte.

The idea that the ancestors of modern cells were RNA cells (ribocytes) can be investigated by asking whether all essential cellular functions might be performed by RNAs. This requires isolating suitable molecules by selection-amplification when the predicted molecules are presently extinct. In fact, RNAs with many properties required during a period in which RNA was the major macromolecular agent in cells (an RNA world) have been selected in modern experiments. There is, accordingly, reason to inquire how such a ribocyte might appear, based on the properties of the RNAs that composed it. Combining the intrinsic qualities of RNA with the fundamental characteristics of selection from randomized sequence pools, one predicts ribocytes with a cell cycle measured (roughly) in weeks. Such cells likely had a rapidly varying genome, composed of many small genetic and catalytic elements made of tens of ribonucleotides. There are substantial arguments that, at the mid-RNA era, a subset of these nucleotides are reproducibly available and resemble the modern four. Such cells are predicted to evolve rapidly. Instead of modifying preexisting genes, ribocytes frequently draw new functions from an internal pool containing zeptomoles (<1 attomole) of predominantly inactive random sequences.

Phenotype↗
PubMed1993

Polymyositis and dermatomyositis: no persistence of enterovirus or encephalomyocarditis virus RNA in muscle.

OBJECTIVES: A persistent infection of enteroviruses and cardioviruses has been implicated in polymyositis and dermatomyositis, but conventional hybridisation studies of the presence of enterovirus RNA and encephalomyocarditis (EMC) virus RNA in affected muscle have yielded conflicting results. To investigate further the possibility of viral persistence, the presence of viral RNA in muscle from patients with adult onset polymyositis and dermatomyositis was investigated using a polymerase chain reaction (PCR) technique. METHODS: Muscle tissue was obtained from 10 patients with polymyositis and five patients with dermatomyositis, all with adult onset active disease. A PCR was performed using primers with high specificity for enterovirus and EMC virus RNA, followed by Southern blot hybridisation with an oligonucleotide probe directed against the internal portion of the amplified product. A PCR directed against the Abelson tyrosine kinase mRNA served as an internal control for the presence and quality of RNA. RESULTS: A specific amplification for enterovirus or for EMC virus could not be seen in any of the muscle biopsy samples, despite a sensitivity of about 30 plaque forming units for enterovirus and of 100 plaque forming units for EMC virus. Southern blot hybridisation confirmed these results in that positive controls hybridised with the oligonucleotide probe, but no signal was obtained with the muscle specimens. CONCLUSION: A sensitive and specific PCR technique showed no evidence of the presence of enterovirus or EMC virus RNA in muscle samples from patients with polymyositis or dermatomyositis. These data do not support the proposal that viral RNA persistence plays a part in these idiopathic inflammatory myopathies.

Adult↗
PubMed2006

Preparation of native and amplified tumour RNA for dendritic cell transfection and generation of in vitro anti-tumour CTL responses.

Recent research indicates that dendritic cells transfected with RNA-encoded tumour-associated antigens (TAA) can generate potent anti-tumour immune responses in vitro and in vivo. RNA is an important source of TAA, but its relatively unstable nature, in addition to often limited availability of tumour tissue, may represent a considerable obstacle for its use. Our first goal was to establish an efficient protocol for the preparation of high quality total RNA from tumour samples. This should then be used as such or be pre-amplified for DC transfection. Therefore native total RNA was prepared from stabilised tissue samples obtained from liver metastases of colon cancer using either solution- or silicagel-based protocols for RNA isolation. The first isolation protocol yielded higher amounts of total RNA, but with lower purity as compared to the second one. No degradation of RNA was observed regardless of the protocol used. Subsequently, we focused on the amplification of mRNA. The fidelity of the amplified mRNA was confirmed by RT-PCR for glyceraldehyde-3-phosphate-dehydrogenase (GADPH) and carcinoembryonic antigen (CEA) coding sequences. We found no differences in the induction of CEA-specific CTL responses between native and amplified RNA-transfected DCs. Additionally, we tested the induction of CTL responses and found that DCs transfected with amplified mRNA originating from either tumour tissue or a cell line were able to induce strong anti-tumour CTL responses in vitro. They were comparable to those induced by native total RNA-transfected DCs. Our results therefore indicate that the amplified mRNA is equivalent to the native one in the induction of anti-tumour CTL responses and can be used for generation of RNA-transfected DCs.

Cell Line, Tumor↗
PubMed2005

Expression of NASG gene and its role in human nasopharyngeal homogenous tissue cells.

BACKGROUND: The NASG gene has been confirmed as a tumor-suppressor gene candidate related to nasopharyngeal carcinoma (NPC) by previous studies. We further investigated the expression and the role of NASG in the homogeneous tissue cells by microdissecting the samples of tissue from human NPC, and introduced a new way to study the expression of specific genes in tumor tissue. METHODS: The RNAlater reagent was used to preserve the samples of tissue from the nasopharynx of NPC patients. The samples were microdissected to harvest the homogeneous tissue cells and then total RNA was isolated from them. The antisense RNA (aRNA) was amplified from the total RNA by "in vitro transcription (IVT)". We investigated NASG expression in the homogeneous tumor cells of NPC (22 samples) and compared it with that in the pure epithelial pillar cells of normal nasopharyngeal (10 samples) by semi-quantitative reverse transcription-polymerase chain reaction (sqRT-PCR). RESULTS: The high quality total RNA could be harvested from the microdissected homogeneous tissue cells of the nasopharynx, then sufficient aRNA was derived from it. NASG gene expression was identified using aRNA by sqRT-PCR and showed that there was significant difference between the average value of case groups and that of control group (t = -5.275, df = 30, P < 0.001). The NASG gene in the subgroups WHOII tended to express lower levels than those in the subgroup WHOIII although this difference was not statistically significant (t = -1.584, df = 20, P = 0.129 > 0.05). CONCLUSIONS: Microdissection was an effective method to obtain the homogeneous tissue cells of nasopharyngeal tissue (including the samples of NPC and non-NPC) in our study. Sufficient aRNA from amplifying total RNA could be used in sqRT-PCR to analyse the expression of NASG in the pure tissue cells. NASG should be a tumor-suppression gene candidate regarding to NPC.

Adult↗
PubMed2004

Application of RT-PCR to clinical diagnosis of micrometastasis of colorectal cancer: A translational research study.

We previously reported in a retrospective study that CEA-based RT-PCR was useful for predicting the prognosis of patients with node-negative colorectal cancer. RT-PCR is well established for laboratory use, but many issues remain to be resolved prior to its clinical application. In addition to the false positive rate of RT-PCR, we addressed several issues, including the timing of lymph node sampling, stability of RNA after surgery, and reproducibility of results. After appropriate modification, including development of a tissue sampling kit, a multi-institutional clinical study was commenced prospectively from November 2001, and 100 patients were enrolled for examination of micrometastasis. RNA was stable in lymph nodes for up to 3 h after surgical resection. This range of sampling time was acceptable to the surgeons. RNA was well preserved in RNA later at -20 degrees C for 3 weeks. Dilutions of MKN45 and LoVo cells served as positive controls for conventional PCR since these controls were found to be highly stable and ensured reproducibility. Moreover, simultaneous use of quantitative PCR (Light Cycler) ensured double confirmation of the results. Our clinical study showed that the quality of RNA was excellent or good in most samples (98 of 100; 98%). Twenty-four of 98 (24.5%) cases were judged to be micrometastasis-positive. In conclusion, the current translational research study established a clinically feasible RT-PCR system for micrometastasis. Our system could potentially be useful as a clinical tool.

Colorectal Neoplasms↗
PubMed1996

Use of the polymerase chain reaction technique to determine c-myc expression in follicular center cell lymphoma.

We determined, in a semiquantitative fashion, the level of expression of c-myc in 18 follicular center cell lymphomas and five non-neoplastic lymph nodes using reverse transcription and the polymerase chain reaction technique (RT-PCR). With this method, RNA is extracted from lymph node specimens and reverse-transcribed to produce cDNA, which is then amplified using primers specific for c-myc sequences that span introns, thus precluding amplification of genomic DNA. Amplified products are compared with beta2-microglobulin sequences co-amplified in each case as a control for the quality of RNA extracted, RT, and PCR amplification. Using cell lines derived from Burkitt's lymphomas, the RT-PCR method yielded results equivalent to standard Northern blot analysis yet required smaller quantities of tissue. The c-myc transcripts were detected in all lymphoma cases studied (seven high, 11 low expression) and in all non-neoplastic lymph nodes. High or low c-myc expression was based on comparison with non-neoplastic lymph nodes. We conclude that the RT-PCR method is a sensitive, reliable method for measuring gene expression in lymphoma tissues and may be useful for studying the role of c-myc in follicular lymphomas.

Adult↗
PubMed2005

A qualitative assessment of direct-labeled cDNA products prior to microarray analysis.

BACKGROUND: The success of the microarray process in determining differential gene expression of thousands of genes is dependent upon the quality and integrity of the starting RNA, this being particularly true of direct labeling via a reverse transcription procedure. Furthermore, an RNA of reasonable quality still may not yield reliable hybridization data if the labeling efficiency was poor. RESULTS: Here we present a novel assay for assessing the quality of directly labeled fluorescent cDNA prior to microarray hybridization utilizing the Agilent 2100 Bioanalyzer, which employs microfluidic technology for the analysis of nucleic acids and proteins. Using varying amounts of RNase to simulate RNA degradation, we show the strength of this un-advertised assay in determining the relative amounts of cDNA obtained from a direct labeling reaction. CONCLUSION: Utilization of this method in the lab will help to prevent the costly mistake of hybridizing poor quality direct labeled products to expensive arrays.

Computational Biology↗
PubMed2005

Protein/RNA coextraction and small two-dimensional polyacrylamide gel electrophoresis for proteomic/gene expression analysis of renal cancer biopsies.

A small amount of bioptic tissue ( approximately 5-10mg of fresh tissue) usually does not contain enough material to extract protein and RNA separately, to obtain preparative two-dimensional polyacrylamide gel electrophoresis (2-DE), and to identify a large number of separated proteins by MS. We tested a method, on small renal cancer specimens, for the coextraction of protein and RNA coupled with 2-DE and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) or quadrupole time-of-flight (Q-TOF) analysis. We coextracted 0.28+/-0.05mg of proteins and 2.5+/-0.33microg of RNA for each 10mg of renal carcinoma tissue. Small and large 2-DE gels were compared: they showed a similar number of spots, and it was possible to match each other; using small format gels, one-fifth of the protein amount was required to identify, by Q-TOF analysis, the same number of proteins identifiable in large-format gel using MALDI-TOF analysis. Quality of RNA coextracted with the proteins was tested by real-time PCR on a set of housekeeping genes. They were quantified with high amplification efficiency and specificity. In conclusion, using 5 to 10mg of fresh tissue, it was possible to perform comprehensive parallel proteomic and genomic analysis by high-resolution, small-format 2-DE gels, allowing approximately 300 proteins identification and 1000 genes expression analysis.

Carcinoma, Renal Cell↗
PubMedDate not supplied

Identification of various exon combinations of the ews/fli1 translocation: an optimized RT-PCR method for paraffin embedded tissue -- a report by the CWS-study group.

BACKGROUND: Chromosomal translocations t(11;22) (q24;q12) are characteristic of about 80-90 % of Ewing's sarcoma family of tumors [bone and soft tissue Ewing's sarcoma and peripheral neuroectodermal tumors (PNET)]. They generate ews/fli1 rearrangements showing great diversity in breakpoint exon combination. In about 5 % of Ewing's tumors, ews is fused to the erg gene at 21q22. The various chimeric proteins encoded may function as aberrant oncogenic transcription factors. These specific translocations can be used for exact molecular diagnosis in these poorly differentiated small round-cell tumors. Moreover, the prognostic relevance of different translocational variants has been previously suggested. Furthermore, the sensitive molecular detection of minimal metastatic and residual disease and its clinical significance can be evaluated. To address these questions more definitively in the large number of patients registered in multicenter studies, it is often necessary to access archival paraffin-embedded tumor tissue if no fresh or frozen tumor material is available for analysis by RT (reverse transcription)-PCR. Specific problems arise from formalin-fixed and paraffin-embedded tissue due to the degradation of RNA and insufficient extraction efficiency. Therefore, primer distance and product size are limited for successful PCR amplification. This conflicts with the requirement for identification of various possible exon combinations by PCR simultaneously using one single primer pair with larger distance. PATIENTS: We examined paraffin embedded soft part tumor tissue samples from 47 Ewing's tumor patients. Patients were treated according to either CWS (Cooperative Weichteilsarkomstudie, CWS-91 or CWS-96) or Euro-E.W.I.N.G. 99 therapy protocols. METHOD: We established a novel RT-PCR method, using 3 different exon specific sets of PCR primer pairs, selected according to the coding ews and fli1 nucleotide sequences (NCBI database), suitable for RT-PCR identification of variant ews/fli1 fusion transcripts in RNA isolated from formalin-fixed, paraffin-embedded tissue. For use in combination with ews -primer, an erg specific primer was selected to alternatively test for ews/erg fusion transcripts. As positive control for the integrity of isolated mRNA, we used the ubiquitously expressed gapdh transcript for RT-PCR amplification in each sample. RESULTS: In 31 cases (= 66 %) of 47 paraffin samples of Ewing's tumors analysed, gapdh control indicated adequate quality of RNA. In 16 cases no gapdh control fragment was amplifiable, nevertheless in 2 of these 16 samples distinct ews fusion products could be detected. In 23 cases we identified ews fusion transcripts. Thereof in 65 % ews exon 7 being fused to fli1 exon 6 (fusion type I), in 22 % to fli1 exon 5 (fusion type II). In 4 % each ews exon 10 being juxtaposed to fli1 either exon 6 or exon 5, respectively. An ews/erg fusion was detected in 4 % ( ews exon 7 fused to erg exon 6). In 10 samples, a gapdh fragment was amplified, but no ews/fli1 or - erg fusion transcript could be identified. The reference pathological review (I. L., Kiel, Germany) disproved the primary histopathology in 5 cases. CONCLUSIONS: Using our different sets of exon specific primer pairs, it was possible to detect 4 different breakpoints of ews/fli1 fusion transcripts and the ews/erg fusion by RT-PCR in RNA isolates from formalin-fixed, paraffin-embedded Ewing's tumor tissue. This method can be a very useful alternative in clinical situations (to ensure diagnosis and perform minimal metastatic and residual disease investigations) and in order to assess prognostic significance of translocation subtypes when no fresh tumor tissue is available.

Electrophoresis, Agar Gel↗
PubMed2027

Workflow for Long-Read Amplicon Sequencing of Chikungunya Virus Using Oxford Nanopore Technology.

This protocol provides a comprehensive, step-by-step workflow for whole-genome sequencing of Chikungunya virus (CHIKV) using an amplicon-based strategy optimized for Oxford Nanopore Technologies (ONT) platforms. The procedure includes detailed instructions for sample handling, viral RNA extraction, quality control, cDNA synthesis, multiplex PCR amplification, library preparation, sequencing, and primary bioinformatic processing. The protocol is designed to maximize reproducibility across laboratories and is suitable for genomic surveillance applications, including outbreak investigation and molecular epidemiology, even when working with low-to-moderate viral loads.

Chikungunya virus↗
PubMed2006

TaqMan PCR assay in the control of RNA normalization in human post-mortem brain tissue.

The brain tissue obtained after death is subjected to several circumstances that can affect RNA integrity. The present study has been directed to reveal possible pitfalls and to control RNA normalization in post-mortem samples in order to recognize the limitations and minimize errors when using TaqMan PCR technology. This has been carried out in samples of the frontal cortex in a series of control and diseased cases covering Parkinson's disease, dementia with Lewy bodies pure form and common form, and Alzheimer's disease. Special attention has been paid to the value of the agonal state, post-mortem delay and pH of the nervous tissue as approximate predictors of the quality of RNA, as well as to the use of the Bioanalyzer to confirm RNA preservation. In addition, since possible disease-modified mRNAs have to be normalized with ideal unaltered RNAs, TaqMan human endogenous control plates have been used to determine the endogenous control most appropriate for the study. beta-glucuronidase (GUS) and beta-actin were good endogenous controls because their expression levels showed a small variation across a representative number of control and pathological cases. RNA stability was also analysed in a paradigm mimicking cumulative delay in tissue processing. GUS mRNA levels were not modified although beta-actin mRNA levels showed degradation at 22 h. Finally, the control of RNA degradation for the normalization of genes of interest was also tested. mRNA expression levels for superoxide dismutase 1 (SOD1) and metalloproteinase domain 22 (ADAM22) were examined at several artificial post-mortem times, and their expression levels compared with those for putative controls beta-actin and GUS. In our paradigm, the expressions of SOD1 and ADAM22 were apparently not modified when normalized with beta-actin. Yet their expression levels were reduced with post-mortem delay when values were normalized with GUS. Taken together, these observations point to practical consequences in TaqMan PCR studies. Short post-mortem delays and acceptable pH of the brain are not sufficient to rule out RNA degradation. The selection of adequate endogenous controls is pivotal in the study. beta-actin and GUS are found to be good endogenous controls in these pathologies, although GUS but not beta-actin expression levels are preserved in samples with long post-mortem delay.

Aged↗
PubMed2005

P-RnaPredict--a parallel evolutionary algorithm for RNA folding: effects of pseudorandom number quality.

This paper presents a fully parallel version of RnaPredict, a genetic algorithm (GA) for RNA secondary structure prediction. The research presented here builds on previous work and examines the impact of three different pseudorandom number generators (PRNGs) on the GA's performance. The three generators tested are the C standard library PRNG RAND, a parallelized multiplicative congruential generator (MCG), and a parallelized Mersenne Twister (MT). A fully parallel version of RnaPredict using the Message Passing Interface (MPI) was implemented on a 128-node Beowulf cluster. The PRNG comparison tests were performed with known structures whose sequences are 118, 122, 468, 543, and 556 nucleotides in length. The effects of the PRNGs are investigated and the predicted structures are compared to known structures. Results indicate that P-RnaPredict demonstrated good prediction accuracy, particularly so for shorter sequences.

Algorithms↗
PubMed2003

Liquid-based pap smears as a source of RNA for gene expression analysis.

The Papanicolaou smear has contributed to a decrease in cervical cancer rates in populations that receive regular screening. However, treatment of women with mildly abnormal cells is problematic because the majority of these women do not develop neoplasia. Thus, new techniques for identification of truly precancerous cells are needed. Characterization of cellular gene expression patterns is now possible through microarray techniques that survey the expression of large numbers of genes simultaneously. Here we have assessed the feasibility of combining new microscopic and molecular technologies to determine gene expression patterns in cervical intraepithelial neoplasia grade 3 cells recovered from liquid cytology-based Papanicolaou smear slides. Laser capture microdissection was used to retrieve cervical cells from ThinPrep prepared slides. The quality of RNA recovered from these cells proved suitable for reverse transcription polymerase chain reaction and for T7 RNA polymerase-based linear amplification of messenger RNA. We developed an optimized RNA amplification protocol that permitted microarray gene expression profiling in samples of as few as 20 cervical cells. This approach combining laser capture microdissection, linear RNA amplification, and microarray gene expression analysis will enable comparison of gene expression patterns between cytologically abnormal and normal cells taken from a single slide and may assist in the differential diagnosis of histologically difficult cases.

Biomarkers↗
PubMed2002

Tornado extraction: a method to enrich and purify RNA from the nephrogenic zone of the neonatal rat kidney.

BACKGROUND: Development of the kidney is a complicated and tightly regulated process. Although several genes responsible for the renal development have been identified to date, the precise mechanisms of spatial and temporal regulation remain to be elucidated. Therefore, expanding our knowledge of molecules that are associated with nephrogenesis will be helpful to understand the whole process. METHODS: To extract RNA selectively from the nephrogenic zone of the developing kidney, we developed a simple and reliable method. RESULTS: This method, named "tornado extraction," enriched RNA of the nephrogenic zone by about 30-fold. In combination with the suppression subtractive hybridization, a considerable number of genes that were differentially expressed in the nephrogenic zone were obtained. These genes included a series of endodermal markers such as albumin and alpha-fetoprotein as well as GDNF (glia-derived neurotrophic factor), osteoblast-specific factor-2 (OSF-2)/periostin and fetuin (one of the major serum proteins in the fetus). CONCLUSION: Tornado extraction has great value in studying genes in the nephrogenic zone of the developing kidney. Since the quality of RNA obtained by this method is excellent, tornado extraction is suitable in combination with other techniques including the subtractive hybridization method and DNA microarray analysis.

Animals↗
PubMed2005

Protecting RNA in fixed tissue: an alternative method for LCM users.

RNA degradation is a major drawback in most common fixation protocols in techniques that require both RNA integrity and preserved morphology, such as laser capture microdissection (LCM) followed by RT-PCR. Moreover, RNA isolation kits especially developed for LCM samples are very expensive. Our aim was to determine an easy protocol that ideally must provide an acceptable morphology, allow proper laser capture of selected cells and improve RNA yield and quality. In this study, retinas were dissected, briefly incubated in a RNA preservative and fixed in 2% paraformaldehyde before being cut on a cryostat. LCM was carried out in retinal sections for immediate RNA isolation, by using TRIzol common protocol with minor modifications. Real-time PCR was performed next in order to compare availability of RNA from samples submitted to different protocols. The use of the RNA preservative followed by a fast fixation did not jeopardize tissue morphology, allowing microdissection of selected cells, combined to minor modifications in usual RNA isolation procedures, significantly improved RNA yield and quality. Furthermore, only LCM samples submitted to our protocol provided amplifiable mRNA, as determined by real-time PCR. Taken together, the combination of the described procedures resulted in a reliable alternative for LCM users.

Animals↗
PubMed2006

Genomic splice-site analysis reveals frequent alternative splicing close to the dominant splice site.

Alternative pre-mRNA splicing may be the most efficient and widespread mechanism to generate multiple protein isoforms from single genes. Here, we describe the genomic analysis of one of the most frequent types of alternative pre-mRNA splicing, alternative 5'- and 3'-splice-site selection. Using an EST-based alternative splicing database recording >47,000 alternative splicing events, we determined the frequency and location of alternative 5'- and 3'-splice sites within the human genome. The most common alternative splice sites used in the human genome are located within 6 nucleotides (nt) of the dominant splice site. We show that the EST database overrepresents alternative splicing events that maintain the reading frame, thus supporting the concept that RNA quality-control steps ensure that mRNAs that encode for potentially harmful protein products are destroyed and do not serve as templates for translation. The most frequent location for alternative 5'-splice sites is 4 nt upstream or downstream from the dominant splice site. Sequence analysis suggests that this preference is a consequence of the U1 snRNP binding sequence at the 5'-splice site, which frequently contains a GU dinucleotide 4 nt downstream from the dominant splice site. Surprisingly, approximately 50% of duplicated 3'-YAG splice junctions are subject to alternative splicing. This high probability of alternative 3'-splice-site activation in close proximity of the dominant 3'-splice site suggests that the second step of the splicing may be prone to violate splicing fidelity.

Alternative Splicing↗