Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “RNA quality”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 109 records · Page 6Linked to original sources
PubMed2006

Gene expression analysis of distinct populations of cells isolated from mouse and human inner ear FFPE tissue using laser capture microdissection--a technical report based on preliminary findings.

Laser Capture Microdissection (LCM) allows microscopic procurement of specific cell types from tissue sections that can then be used for gene expression analysis. We first tested this method with sections of adult mouse inner ears and subsequently applied it to human inner ear sections. The morphology of the various cell types within the inner ear is well preserved in formalin fixed paraffin embedded (FFPE) sections, making it easier to identify cell types and their boundaries. Recovery of good quality RNA from FFPE sections can be challenging, however, recent studies in cancer research demonstrated that it is possible to carry out gene expression analysis of FFPE material. Thus, a method developed using mouse FFPE tissue can be applied to human archival temporal bones. This is important because the majority of human temporal bone banks have specimens preserved in formalin and a technique for retrospective analysis of human archival ear tissue is needed. We used mouse FFPE inner ear sections to procure distinct populations of cells from the various functional domains (organ of Corti, spiral ganglion, etc.) by LCM. RNA was extracted from captured cells, amplified, and assessed for quality. Expression of selected genes was tested by RT-PCR. In addition to housekeeping genes, we were able to detect cell type specific markers, such as Myosin 7a, p27(kip1) and neurofilament gene transcripts that confirmed the likely composition of cells in the sample. We also tested the method described above on FFPE sections from human crista ampullaris. These sections were approximately a year old. Populations of cells from the epithelium and stroma were collected and analyzed independently for gene expression. The method described here has potential use in many areas of hearing research. For example, following exposure to noise, ototoxic drugs or age, it would be highly desirable to analyze gene expression profiles of selected populations of cells within the organ of Corti or spiral ganglion cells rather than a mixed population of cells from whole inner ear tissue. Also, this method can be applied for analysis of human archival ear tissue.

Animals↗
PubMed2003

A rapid and effective method of extracting fully intact RNA from thermophilic geobacilli that is suitable for gene expression analysis.

Extraction of intact RNA is essential for quantitative gene expression analysis. Isolating high quality RNA from gram-positive bacteria is known to be problematic particularly from organisms that have optimal growth temperatures greater than 45 degrees C. We report a novel extraction protocol for the rapid isolation of fully intact RNA from thermophilic Geobacillus thermoleovorans using a lysing matrix containing a mixture of ceramic and glass beads, triisopropylnaphthalene sulfonic acid (TNS), and p-4-aminosalicyclic acid (PAS). Combining both detergents, TNS and PAS, appeared to increase denaturation of RNases at thermophilic temperatures. Gel electrophoresis revealed that only RNA isolated using the TNS-PAS procedure demonstrated sharp, undegraded 23S, 16S, and 5S ribosomal RNA bands. RNA extracted from geobacilli using commercially available kits was extensively degraded and was not suitable for detecting gene expression. Total RNA yields extracted with the TNS-PAS protocol were greater than eightfold higher than those obtained with available kits. Critically, it was also shown that only RNA isolated with the TNS-PAS-based method was suitable for monitoring thermophile gene expression patterns using RT-PCR analysis.

Aminosalicylic Acid↗
PubMed2006

The use of needle biopsy for assessment of placental gene expression.

OBJECTIVE: The purpose of this study was to test the hypothesis that placental samples that are obtained by needle aspiration ex vivo are useful for the determination of villus gene expression. STUDY DESIGN: Placental biopsy was performed with a spinal needle after uncomplicated deliveries. Villi were inspected microscopically, and RNA was extracted and analyzed with capillary electrophoresis. Gene expression was determined with quantitative polymerase chain reaction. RESULTS: We obtained more placental villous fragments per aspiration using a 20-gauge needle (5.2 +/- 1.8 fragments) than with a 22-gauge needle (3.3 +/- 1.6 fragments; P < .01). RNA quality was adequate, based on the 28S and 18S recombinant RNA bands, with a mean 260/280 ratio of 1.88. The amount of extracted RNA correlated with the number of villous fragments per aspirate. Importantly, the expression of NDRG1 and hPL, both markedly altered in hypoxia, was consistent between villi that were obtained by either needle or standard biopsy. CONCLUSION: Placental samples that are obtained by ex vivo needle aspiration are useful for the extraction of RNA and for the determination of villous gene expression.

Biopsy, Needle↗
PubMed2005

Intervening sequence acquired by lateral gene transfer in Tropheryma whipplei results in 23S rRNA fragmentation.

Completion of Tropheryma whipplei genome sequencing may provide insights into the evolution of the molecular mechanisms underlying the pathogenicity of this microorganism. The first postgenomic application was the successful design of a comprehensive culture medium that allows axenic growth of this bacterium, which is particularly recalcitrant to cultivation. This achievement in turn permitted analysis of T. whipplei RNA without contaminating eukaryotic nucleic acids. To obtain high-quality RNA, several extraction methods were compared, but under all conditions tested an atypical profile was observed. By using a Northern blot assay we demonstrated that an insertion sequence previously described in T. whipplei 23S rRNA is in fact an intervening sequence excised during maturation. This cleavage could involve an RNase III identified in the genome of this microorganism. Among the bacteria with a 23S rRNA insertion sequence, T. whipplei is the only gram-positive microorganism. We present phylogenetic evidence that this mobile genetic element was acquired by lateral gene transfer from another enteric bacterium.

Actinomycetales↗
PubMed2004

A general method for rapid and nondenaturing purification of RNAs.

A key bottleneck in RNA structural studies is preparing milligram quantities of RNA, and current techniques have changed little in over a decade. To address this, we have developed an affinity tag-based purification method of RNA oligonucleotides. The tag is attached to the 3'-end of almost any desired RNA sequence, allowing for the rapid and specific removal of the RNA of interest directly from in vitro transcription reactions using an affinity column to which a specific RNA-binding protein has been attached. Following a wash, the RNA of interest is eluted by the addition of imidazole to the column, activating a mutant HdeltaV ribozyme incorporated into the tag. The affinity column can then be rapidly regenerated using conditions that release the protein-RNA tag interaction without denaturing the protein. To demonstrate that this method rapidly generates high-quality RNA, we have transcribed, purified, and generated diffraction-quality crystals of a mutant form of the Tetrahymena thermophila P4-P6 domain in a 48-h time period.

Affinity Labels↗
PubMed1993

Application of dual-digitonin-pulse perfusion to the study of hepatic mRNA zonation.

Heterogeneous zonation of hepatic protein expression over the liver lobule has been recognized by using several analytical techniques, including microdissection, selective cell isolation, immunohistochemistry and hybridization of mRNA in situ. We previously employed the technique of dual-digitonin-pulse perfusion for the highly selective collection and analysis of periportal and perivenous soluble protein. In the present work we have now documented the feasibility of the application of this technique to the study of zonal distribution of mRNA. By using a split-stream design, both protein and RNA fractions can be simultaneously collected from hepatic zones. High-quality RNA (average yield approximately 9-33 micrograms of total RNA per mg of eluted protein) is obtained for analysis. As analysed by immunoblotting and Northern-blot analysis, the zonal distribution of several important cytosolic metabolic enzymes and their mRNAs can be documented. This technique is also applicable to the study of mRNAs for organelle- and membrane-associated proteins that are not recoverable with this digitonin-lysis technique. The application of this experimental technique should allow further molecular insight into the mechanisms underlying zonation of hepatic function.

Animals↗
PubMed2004

Systematic analysis of T7 RNA polymerase based in vitro linear RNA amplification for use in microarray experiments.

BACKGROUND: The requirement of a large amount of high-quality RNA is a major limiting factor for microarray experiments using biopsies. An average microarray experiment requires 10-100 microg of RNA. However, due to their small size, most biopsies do not yield this amount. Several different approaches for RNA amplification in vitro have been described and applied for microarray studies. In most of these, systematic analyses of the potential bias introduced by the enzymatic modifications are lacking. RESULTS: We examined the sources of error introduced by the T7 RNA polymerase based RNA amplification method through hybridisation studies on microarrays and performed statistical analysis of the parameters that need to be evaluated prior to routine laboratory use. The results demonstrate that amplification of the RNA has no systematic influence on the outcome of the microarray experiment. Although variations in differential expression between amplified and total RNA hybridisations can be observed, RNA amplification is reproducible, and there is no evidence that it introduces a large systematic bias. CONCLUSIONS: Our results underline the utility of the T7 based RNA amplification for use in microarray experiments provided that all samples under study are equally treated.

Adenocarcinoma↗
PubMed2002

Postmortem RNA and protein stability in perinatal human lungs.

The availability of fetal and neonatal lung tissue is an invaluable resource to elucidate the molecular regulation of human lung development. In this study, we have investigated the mRNA and protein stability of perinatal lung tissues treated with RNA (Ambion Inc., Austin, TX) or snap frozen in liquid nitrogen (LN ). Lung samples were obtained from 25 consecutive perinatal autopsies of live-born and stillborn infants (median gestational age, 23 weeks) with various clinical presentations. Treatment of lung tissue with RNA yielded more total RNA and protein than LN freezing. The integrity of RNA, assessed by spectrophotometry and gel electrophoresis, was equivalent between both tissue preservation methods, and both methods produced RNA suitable for reverse transcriptase-polymerase chain reaction analysis of representative genes (beta-actin and surfactant protein-B [SP-B]). Similarly, the protein integrity of RNA -treated tissues was equivalent to that of LN -frozen tissues, as judged by Western blot analysis of SP-B/actin protein expression. Although the total yield was similar in live-born, nonmacerated stillborn and macerated stillborn infants, only RNA and protein from live-born or nonmacerated stillborn infants was suitable for subsequent molecular analyses. Within the 41-hour range studied, the duration of the postmortem interval did not affect the yield or integrity of RNA and protein with either tissue preservation method. In summary, high-quality RNA and protein, suitable for routine molecular analyses, can be obtained from postmortem lung tissue from live-born and nonmacerated stillborn infants, even with prolonged postmortem intervals. RNA is equivalent, if not superior, to LN for preservation of postmortem RNA and protein in developing human lungs.

Autolysis↗
PubMed1990

Bacterial RNA isolation with one hour centrifugation in a table-top ultracentrifuge.

A procedure for the rapid preparation of cesium-chloride purified RNA from E. coli and the cyanobacterium Synechococcus sp. PCC7942 is described. Cells are lysed in modified sucrose, Triton X-100, EDTA, Tris buffer with phenol/chloroform. The cleared lysate is extracted further with phenol/chloroform and RNA is peleted by centrifugation through a 5.7 M CsCl cushion. High quality RNA can be prepared in three hours using this procedure.

Aurintricarboxylic Acid↗
PubMed2001

Gene expression in a pure population of odontoblasts isolated by laser-capture microdissection.

Studies of odontoblast differentiation and function have been limited due to difficulties in obtaining sufficient numbers of intact cells. We describe a novel approach of laser-capture microdissection to obtain homogenous populations of pre-odontoblasts and odontoblasts from tissue sections of mouse molar cusp tips. Fixation, processing, and staining conditions were assessed for the optimal retrieval of total RNA from microdissected odontoblasts. Fluorometric assays and RT-PCR analysis of alpha1(I) collagen, dentin sialophosphoprotein (Dspp), and osteocalcin (OC) confirmed that the total RNA from three-day-old captured odontoblasts was sufficient in quantity and quality. Odontoblast-specific gene expression was studied by RT-PCR analysis performed in a single streptavidin-coated tube. At E15.5, Days 0 and 3, gene expression in laser-captured odontoblasts resembled that seen in vivo by in situ hybridization. The use of LCM is thus a valuable means of retrieving quality RNA from discrete populations of odontoblasts at different stages of dentinogenesis.

Animals↗
PubMed2006

Transcriptional profiling of degraded RNA in cryopreserved and fixed tissue samples obtained at autopsy.

BACKGROUND: Traditional multiplexed gene expression methods require well preserved, intact RNA. Such specimens are difficult to acquire in clinical practice where formalin fixation is the standard procedure for processing tissue. Even when special handling methods are used to obtain frozen tissue, there may be RNA degradation; for example autopsy samples where degradation occurs both pre-mortem and during the interval between death and cryopreservation. Although specimens with partially degraded RNA can be analyzed by qRT-PCR, these analyses can only be done individually or at low levels of multiplexing and are laborious and expensive to run for large numbers of RNA targets. METHODS: We evaluated the ability of the cDNA-mediated Annealing, Selection, extension, and Ligation (DASL) assay to provide highly multiplexed analyses of cryopreserved and formalin fixed, paraffin embedded (FFPE) tissues obtained at autopsy. Each assay provides data on 1536 targets, and can be performed on specimens with RNA fragments as small as 60 bp. RESULTS: The DASL performed accurately and consistently with cryopreserved RNA obtained at autopsy as well as with RNA extracted from formalin-fixed paraffin embedded tissue that had a cryopreserved mirror image specimen with high quality RNA. In FFPE tissue where the cryopreserved mirror image specimen was of low quality the assay performed reproducibly on some but not all specimens. CONCLUSION: The DASL assay provides reproducible results from cryopreserved specimens and many FFPE specimens obtained at autopsy. Gene expression analyses of these specimens may be especially valuable for the study of non-cancer endpoints, where surgical specimens are rarely available.

Journal Article↗
PubMed2005

Effects of RNA degradation on gene expression analysis of human postmortem tissues.

The Affymetrix GeneChip platform was used to build a gene expression database of the normal human body. Postmortem human tissues represent a valuable source of biological materials for this type of study, but their use entails some delays before harvesting such tissues. We first evaluated the RNA quality obtained from tissues obtained 3-5 h postmortem and found variations that were both tissue and donor-dependent. RNAs extracted from brain regions were of higher quality than those obtained from the gut, while the cause of death was a significant factor in donor-dependent differences. To avoid these variables, we used rat duodenum to determine the effects of RNA degradation on the analysis of gene expression. Surprisingly, even samples exhibiting significant RNA degradation yielded robust gene expression results, comparable to those obtained using intact samples at a certain signal intensity cutoff. We extended these findings to our human expression database and obtained similar results, indicating that the Affymetrix platform, which is biased to the 3' end of transcripts for detection, can tolerate significant RNA degradation, while still yielding high quality expression data. Our resulting body index expression database is a valuable research tool. As examples of potential uses, we report novel expression sites for four potential therapeutic targets--CCL27, GPR22, GPR113 and GPR128--and as well as a set of thymus-specific genes, including three not previously associated with the thymus.

Animals↗
PubMed2006

Quality assessment of Affymetrix GeneChip data.

Affymetrix GeneChips are one of the best established microarray platforms. This powerful technique allows users to measure the expression of thousands of genes simultaneously. However, a microarray experiment is a sophisticated and time consuming endeavor with many potential sources of unwanted variation that could compromise the results if left uncontrolled. Increasing data volume and data complexity have triggered growing concern and awareness of the importance of assessing the quality of generated microarray data. In this review, we give an overview of current methods and software tools for quality assessment of Affymetrix GeneChip data. We focus on quality metrics, diagnostic plots, probe-level methods, pseudo-images, and classification methods to identify corrupted chips. We also describe RNA quality assessment methods which play an important role in challenging RNA sources like formalin embedded biopsies, laser-micro dissected samples, or single cells. No wet-lab methods are discussed in this paper.

Animals↗
PubMed2006

Extraction of high-integrity RNA suitable for microarray gene expression analysis from long-term stored human thyroid tissues.

INTRODUCTION: Isolation of high-quality RNA from fresh-frozen thyroid tissues stored for more than a decade would open novel options for gene expression profiling. Herein, we describe successful extraction of high-integrity RNA from human thyroid tissues that were stored for more than a decade. METHODS: Seventy-nine samples (15 goitres, 20 follicular adenomas, 30 papillary carcinomas, 14 follicular carcinomas) that were shock-frozen in isopentane and stored for a median of 11 years (range 1-16 years) were processed using standard precipitation and column filtration techniques. RNA integrity was assessed by electrophoresis using the RNA integrity number (RIN) algorithm and by gene expression profiling determining the 3'/5' ratio of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene and the percentage of transcripts detected on the Affymetrix U133 2.0 human genome GeneChip. RESULTS: The median RNA yield was 1.9 microg/mg tissue (papillary carcinoma 2.1 microg/mg, range 0.2-7.2 microg/mg; follicular carcinoma 2.4 microg/mg, range 0.2-3.2 microg/mg; goitre 1.4 microg/mg, range 0.1-5.4 microg/mg; follicular adenoma 1.6 microg/mg, range 0.1-6.2 microg/mg; p = 0.46) with an 8.6 (7.3-9.8) median RIN. The median GAPDH gene 3'/5' ratio was 1.43 (1.34-1.52) and the median percentage of present calls was 48.1% (42.7-52.0%). CONCLUSIONS: Age and entity independent RNA suitable for expression profiling can be extracted from long-term stored fresh-frozen human thyroid tissues.

Adenocarcinoma, Follicular↗
PubMed2004

Reactive oxygen species induce RNA damage in human atherosclerosis.

BACKGROUND: Reactive oxygen species (ROS)-induced DNA damage has recently been identified in both human and experimental atherosclerosis. This study was undertaken to investigate whether RNA damage occurs in human atherosclerotic plaques and whether this could be related to oxidative stress. MATERIALS AND METHODS: The integrity of total RNA isolated from carotid endarterectomy specimens (n = 20) and nonatherosclerotic mammary arteries (n = 20) was analyzed using an Agilent 2100 Bioanalyser (Agilent Technologies, Palo Alto, CA). Oxidative modifications of RNA were detected by immunohistochemistry. RESULTS: Eleven out of 20 atherosclerotic plaques showed a significant reduction of the 18S/28S rRNA peaks and a shift in the RNA electropherogram to shorter fragment sizes. In contrast, all mammary arteries showed good-quality RNA with clear 18S and 28S rRNA peaks. Strong nuclear and cytoplasmic immunoreactivity for oxidative damage marker 7,8-dihydro-8-oxo-2'-guanosine (8-oxoG) could be detected in the entire plaque in smooth muscle cells (SMCs), macrophages and endothelial cells, but not in SMCs of adjacent normal media or in mammary arteries. Cytoplasmic 8-oxoG staining in the plaque clearly diminished when tissue sections were pretreated with RNase A, suggesting oxidative base damage of RNA. In vitro treatment of total RNA with ROS-releasing compounds induced RNA degradation. CONCLUSION: Both loss of RNA integrity and 8-oxoG oxidative modifications were found in human atherosclerotic plaques. Because RNA damage may affect in vitro transcript quantification, RT-PCR results must be interpreted cautiously if independent experimental validation (e.g. evaluation of RNA integrity) is lacking.

Arteriosclerosis↗
PubMed2004

Real-time quantitative PCR measurement of thyroglobulin mRNA in peripheral blood of thyroid cancer patients and healthy subjects.

Follow-up of recurrent differentiated thyroid carcinoma involves the measurement of serum thyroglobulin (Tg). However, Tg autoantibodies are present in a high proportion of thyroid carcinoma patients (up to 25%) and these can interfere with the Tg immunoassays. To overcome this obstacle, investigators have used real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) to measure Tg mRNA in the blood of patients with differentiated thyroid cancer, with varying degrees of success. In the present study, we demonstrate the first reported use of the PAXgene Blood RNA collection tube and extraction kit method for the preparation of RT-PCR-quality RNA with subsequent deployment of the latter in the development of a specific, sensitive, and reproducible Taqman assay for the detection and quantification of thyroglobulin mRNA. Beta-actin mRNA was also assayed and results are expressed as a ratio of Tg to beta-actin mRNA. The intra-assay coefficient of variations (CVs) for Tg and beta-actin mRNA assay were 27.7% and 25.4%, respectively. Inter-assay CVs were 20.8% and 28.8%, respectively, for the two assays. Tg mRNA was detected in all cancer subjects (n = 42) and healthy individuals (n = 20). Tg mRNA was significantly higher in cancer patients than in the healthy subjects (0.00169 +/- 0.00013 vs. 0.00051 +/- 0.00015; P<0.0001). Fourteen cancer patients had detectable levels of serum Tg, and Tg mRNA levels tended to be higher in these than in cancer subjects with undetectable serum Tg (0.00188 +/- 0.00021 vs. 0.00157 +/- 0.000178; P = 0.08). Circulatory Tg mRNA measurement may serve a useful role in the assessment of thyroid cancer.

Actins↗
PubMed2005

Gene expression profiling of HIV-1 infection using cDNA microarrays.

To illustrate the methods employed in gene expression profiling using cDNA microarrays, infection of CD4+ T cell lines with HIV-1LAI is used to identify expression changes relevant to in vitro HIV-1 infection. Cell lines are infected at a high multiplicity of infection to ensure a population of near-synchronously infected cells to be compared to uninfected cells. Infection status is verified using flow cytometry to determine the intracellular expression of the viral gag p24 protein before samples are harvested for total RNA extraction. Total RNA is extracted and amplified using commercially available kits, and RNA quality is verified using Bioanalyzer technology. To obtain fluorescently labeled cDNA probes, the amplified RNA is reverse-transcribed to yield cDNA, using random nonamers in the presence of dye-labeled dCTP. After first-strand cDNA synthesis, RNA is degraded and the probes are purified. For each infection condition (LAI and mock), two slides are hybridized with identical probes generated from the same RNAs, but with fluorescent labels reversed on one of the slides to control for dye-specific effects. Troubleshooting strategies and issues to consider prior to starting the experiment are discussed in detail in the notes section.

CD4-Positive T-Lymphocytes↗
PubMed2004

Epithelial cell preparation for proteomic and transcriptomic analysis in human pancreatic tissue.

Standardized sample preparation procedures constitute a prerequisite for obtaining reliable and reproducible results in gene expression research in humans. In particular, in diseases such as pancreatic cancer and pancreatitis, isolating epithelial cells is an important step preceding such research. In pancreatic tissue, the high amount of RNAases is a further problem when it comes to obtaining high-quality RNA, and the presence of secreted proteases accelerates protein degradation. We developed a successful method that addresses these different problems. This method, which uses epithelial cell surface antibody Ber-Ep4, proteases, and RNAases inhibitors, leads to a significant enrichment (> 95% purity) of epithelial cells from fresh human tissue samples and allows for both proteomics (Western Blot, 2D PAGE) and transcriptomics studies (rtPCR, cDNA microarray). Compared with other cell purification procedures, this method is characterized by several advantages: a large quantity of cells available for downstream analysis, combined transcriptomics and proteomics studies using the same samples, better reproducibility of proteomics studies, and an acceptable yield (63%) for gene expression arrays studies. Moreover, a quality control protocol addressing the needs of the industry and the requirements of regulatory agencies is proposed.

Epithelial Cells↗