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PubMed2004

Single-stranded linear amplification protocol results in reproducible and reliable microarray data from nanogram amounts of starting RNA.

The range of scientific questions utilizing DNA microarray techniques is limited by the fact that these methods require 5-40 microg of high-quality total RNA. Thus, methods that reliably amplify the starting RNA amount could expand the applicability of DNA microarray technology. We developed a single-stranded linear amplification protocol (SLAP) that combines the reproducibility of in vitro transcription and the amplification robustness of polymerase chain reactions. We compared SLAP to the NIH-IVT amplification protocol. SLAP displayed excellent conservation of the 5'/3' signal and demonstrated the most robust amplification, producing the recommended amounts of biotin-labeled RNA with as little as 0.002 microg of starting RNA. Both SLAP and NIH-IVT methods demonstrated good reproducibility, but SLAP maintained the highest level of reliability with RNA starting amounts of <0.05 microg. These results suggest that SLAP is an excellent alternative to IVT-based amplification protocols when RNA is limited by small sample size.

Biotin↗

PubMed1986

Extensive postmortem stability of RNA from rat and human brain.

The postmortem stability of brain RNA was measured in rat and human samples for up to 48 hr. Whole rat brains, cooled at a rate approximating that of human brains awaiting autopsy, were collected at intervals from 0 to 48 hr after death and frozen. These samples were compared with freshly obtained, unfrozen rat brains. Two potentially independent characteristics of these RNA populations were measured: the recovery or yield of RNA/gram tissue (quantity) and the integrity or extent of degradation (quality). Total RNA yields were similar after all postmortem delays. Hybridization of [32P]-labeled cDNA probes to nitrocellulose filter blots of electrophoretically separated rat brain RNA failed to reveal degradation of the specific rat brain mRNAs during the postmortem period. Similarly, in vitro translation of these same rat total RNA samples produced high molecular weight translation products with no differences between long and short postmortem times. Human cerebral cortex RNA was prepared by the same methods as those used for rat brain from a neurosurgical sample and four other donors with postmortem intervals from 7 to 36 hr. Typically, the yield of total RNA from human brain was 40-50% of the yield from rat brain. When analyzed by RNA gel blot hybridization studies, as for rat brain RNA, human cortical RNA appeared slightly degraded. However, the degree of apparent RNA degradation was not related to the postmortem interval. In vitro translation products of human cortical RNA revealed high molecular weight peptides at all postmortem intervals, but slightly less [35S]incorporation into these bands was found at the longer postmortem intervals relative to the shorter times. Together, these results demonstrate an extensive stability of brain RNA that invites aggressive use of molecular genetic techniques for the study of human neurodegenerative diseases.

Animals↗

PubMed2004

The use of real-time PCR analysis in a gene expression study of Alzheimer's disease post-mortem brains.

The measurement of gene expressions in brains with neurodegenerative diseases is a major area of brain research. The objective of our research was to determine whether quantitative real-time PCR could measure messenger RNA (mRNA) expression in brains with post-mortem intervals beyond 12h. In the present paper, we examined the quality of RNA from brain specimens of both Alzheimer's disease (AD) patients (n = 13) and non-demented normal control subjects (n = 6). To determine a unregulated endogenous reference gene in AD, we measured mRNA expressions of the commonly used reference genes beta-actin, 18S rRNA, and GAPDH. In addition, we determined whether post-mortem interval, brain weight, or age at death influences mRNA expression. Our real-time PCR analysis results indicate that mRNA expression can be detected in all brain specimens for beta-actin, 18S rRNA, GAPDH, and also synaptophysin, a known marker for AD. Further, using real-time PCR analysis, we found that beta-actin and 18S rRNA are differentially expressed in the brain specimens of both AD and control subjects, while GAPDH is similarly expressed in AD and control brain specimens. These findings suggest that GAPDH can be used as a endogenous reference gene in the study of AD brains. A comparative gene expression analysis also suggests that synaptophysin is down-regulated in AD brain specimens compared to control brain specimens.

Actins↗

PubMed1994

Improvement of hepatitis C virus RNA polymerase chain reaction through a multicentre quality control study. French Study Group for the Standardization of Hepatitis C Virus PCR.

Nine French laboratories, using the polymerase chain reaction (PCR) for detection of hepatitis C virus (HCV) RNA, initiated a quality control study to assess and to improve the specificity and sensitivity of their procedures. The study was carried out in three rounds, based on coded panels consisting of anti-HCV positive and anti-HCV negative samples. For the first panel, each laboratory followed its own protocol: 100% sensitivity was observed in two laboratories, 100% specificity in seven. For the second panel, all laboratories were required to use both their own procedure and a consensus procedure established from those laboratories which provided the best results on the first panel. With their own procedure, 100% sensitivity was observed in five laboratories and 100% specificity in all. With the common procedure, 100% sensitivity was observed in all but one, and 100% specificity in all. The third panel included three positive samples with four successive dilutions. For two samples, 8/8 laboratories had positive signals until the 1/100 dilution and discrepant results beyond this dilution; for the third sample, the signals were more discrepant. These results indicate that optimization and standardization of PCR may help laboratories to improve their procedure.

Base Sequence↗

PubMed2004

Does extraction of DNA and RNA by magnetic fishing work for diverse plant species?

An automated nucleic acid extraction procedure with magnetic particles originally designed for isolation of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) from animal tissues was tested for plant material. We isolated genomic DNA and total RNA from taxonomically diverse plant species representing conifers (Scots pine), broad-leaved trees (silver birch and hybrid aspen), dwarf shrubs (bilberry), and both monocotyledonous (regal lily) and dicotyledonous (Saint John's wort, round-leaved sundew, and tobacco) herbaceous plants. Buffers developed for DNA extraction were successfully used in addition to manufacturer's extraction kits. The quality of RNA was appropriate for many applications, but the quality of DNA was not always sufficient for polymerase chain reaction (PCR) amplification. However, we could strikingly improve the quality by eliminating the adherent compounds during the extraction or later in the PCR phase. Our results show that the use of the procedure could be extended to diverse plant species. This procedure is especially suitable for small sample sizes and for simultaneous processing of many samples enabling large-scale plant applications in population genetics, or in the screening of putative transgenic plants.

Chemical Fractionation↗

PubMed2004

Superior 5' homogeneity of RNA from ATP-initiated transcription under the T7 phi 2.5 promoter.

Transcription from the commonly used GTP- initiating T7 class III promoter phi6.5 frequently produces heterogeneous RNA at both 3' and 5' ends. We demonstrate here that RNA transcripts from the T7 class II promoter phi2.5 have superior 5' homogeneity over those from the phi6.5 promoter, with comparable total RNA yields. The overall homogeneity of RNA transcripts is improved to different degrees depending on RNA sequences, although transcription under phi2.5 does not affect the 3' heterogeneity of RNA. In combination with 3' RNA trimming by DNAzymes or ribozymes, this ATP- initiated transcription system based on the T7 phi2.5 promoter can provide excellent quality of RNA for applications requiring a high degree of RNA size homogeneity.

3' Untranslated Regions↗

PubMed1996

Ensuring recovery of intact RNA from rat pancreas.

The isolation of intact RNA from rat pancreas is compromised by autolysis and by the presence of endogenous ribonucleases. In order to ameliorate recovery we systematically investigated available RNA extraction methods and paid particular attention to the influence of frozen storage and ribonuclease inhibition strategies on overall yield and quality of RNA. Modifications to the basic procedure of Chomczynski and Sacchi (1987) are described which allow, reproducibly, to obtain rat pancreatic RNA suitable for Northern blot hybridization, RT-PCR, and differential display analysis.

Animals↗

PubMed2002

A reliable method for total RNA extraction from frozen human bone marrow samples taken at diagnosis of acute leukaemia.

This report describes a newly developed method using Trizol LS reagent that can reliably extract high quality total RNA from frozen human leukaemic bone marrow samples. Extraction of total RNA from 71 frozen leukaemic bone marrow samples obtained at the time of diagnosis produced a median yield of 145 micro g/ml leukaemic bone marrow. Total RNA samples could be reverse transcribed into cDNA and used successfully in the reverse transcription polymerase chain reaction amplification of B2M transcripts in 68 of 71 cases. A multivariate linear regression analysis revealed that significant predictors of RNA yield were both sample volume (< 1 ml v > 1 ml; p = 0.003) and peripheral blood white cell count (< 5 x 10(9) v >or= 5 x 10(9) white blood cells/litre; p = 0.011). The percentage of blasts present, leukaemia subtype, and sample storage period at -80 degrees C (up to 945 days) were not predictors of total RNA yield. This method of total RNA extraction should be of interest to diagnostic and research staff using frozen bone marrow samples for molecular analyses. Similarly, the lack of association between sample storage period at -80 degrees C and total RNA yield should be of interest to the administrators of tumour banks housing frozen bone marrow samples.

Acute Disease↗

PubMed1997

A novel highly reproducible quantitative competitve RT PCR system.

Small changes in plasma fibrinogen concentration are often clinically important. To detect changes in fibrinogen mRNA level, we have designed a novel quantitative competitive reverse transcriptase (RT) PCR system, based on our recent understanding that an important factor causing imprecision with RT PCR is a change in the initial ratio of target to standard RNA templates during reverse transcription. The system comprises: (1) titration of a fixed amount of standard RNA with four different amounts of total RNA in each tube during cDNA synthesis; (2) amplification of each of the four cDNAs with four consecutive PCR cycles; and (3) quality control reactions in which synthesised quality control RNA (rather than total RNA) is added to the standard RNA under the same RT PCR conditions as the assay reaction. To obtain reproducible data, we suggest three criteria for analysis of RT PCR results. Employing these criteria, reproducibility of RT PCR was markedly improved with an inter-assay CV of 6.5% (n=5). Using this system, we are able to detect reduction in mRNA levels of all fibrinogen genes caused by dietary protein restriction in rats after weaning (alpha 39%, p= 0. 028; beta 55%, p = 0.017; gamma 35 %, p= 0.038), with a parallel reduction of plasma fibrinogen concentration (mean (+/-SD), 157(+/-19) versus 130(+/-14) mg/dl, p=0.006).

Analysis of Variance↗

PubMed1991

Isolation of functional RNA from plant tissues rich in phenolic compounds.

A method for the isolation of RNA from different tissues of trees (seedlings, saplings, and adult trees) is described. Using this procedure it is possible to remove large amounts of disturbing polyphenolic compounds from nucleic acids. The method involves an acetone treatment of the freeze-dried and powdered plant material, the use of high salt concentrations in the extraction buffer and an aqueous two-phase system. These steps were combined with the conventional phenol/chloroform extraction and CsCl centrifugation. The method has been successfully applied to the isolation and purification of RNA from pine (Pinus sylvestris L. and Pinus mugo Turr.), Norway spruce (Picea abies L.), and beech (Fagus sylvatica L.). The functional quality of RNA extracted by this procedure has been characterized by its uv spectrum, by agarose gel electrophoresis with ethidium bromide staining, Northern blot hybridization, and in vitro translation.

Acetone↗

PubMed2004

Comparison of ethanol versus formalin fixation on preservation of histology and RNA in laser capture microdissected brain tissues.

Although RNA can be retrieved from formalin-fixed, paraffin-embedded (FFPE) tissues, the yield is low, and the RNA is fragmented. Recent advances in gene expression profiling underscore the importance of identifying a fixative that preserves histology and mRNA. We demonstrated that, for immersion fixation of brains, 70% ethanol is superior to formalin for mRNA preservation. RNA yield from ethanol-fixed tissues was 70% of the yield from fresh frozen specimens, but only a negligible quantity was recovered from formalin-fixed tissues. RNA from ethanol-fixed brains showed integrity comparable to RNA from fresh frozen tissues, and RT-PCR using RNA from ethanol-fixed tissues was consistently successful. RNA from FFPE tissues composed of low-molecular weight fragments, and their use in RT-PCR failed repeatedly. The yield and quality of RNA from ethanol-fixed brains were unaffected after immersion at 4 degrees C for 2 weeks. In a blinded comparison to FFPE tissues, ethanol-fixed specimens were judged to show comparable histology and superior immunostaining. After laser capture microdissection (LCM), we failed to recover mRNA from FFPE tissues but retrieved mRNA from ethanol-fixed tissues for RT-PCR and cDNA microarray analysis. We conclude that 70% ethanol preserves RNA integrity and is suitable for expression profiling of brain tissues by LCM and cDNA microarray.

Animals↗

PubMed2026

When light colour matters: Spectral quality is associated with distinct small RNA candidates in Arabidopsis thaliana.

The spectral composition of incident light is perceived by plant photoreceptors and can rapidly reshape downstream gene expression programs; however, its impact on the small-RNA layer beyond annotated miRNAs remains incompletely characterized. The objective of this study was to determine whether 3 h exposure of Arabidopsis thaliana rosettes to blue, green, red, or white light at an equal photosynthetic photon flux density (PPFD) of 400 μmol m-2 s-1 is associated with distinct profiles of candidate small RNAs. A stringent discovery and post-processing workflow was applied to identify high-confidence, treatment-associated small RNA candidates beyond annotated miRNA. The miRkwood-based pipeline, combined with additional filtering and contextual annotation, recovered a compact set of candidates dominated by the blue-light treatment (41 candidates), compared with fewer candidates associated with red (8), green (6), and white light (3). Genomic-context analysis indicated that most candidate sites were intergenic, with candidates detected under green light being entirely intergenic, and overlap with transposon annotations was used to distinguish candidates arising from transposon-rich genomic space. Sequence-feature profiling revealed pronounced treatment-dependent terminal nucleotide biases at both 5' and 3' ends, consistent with spectrum-associated shifts in length-class and terminal-nucleotide signatures that are informative for AGO-loading hypotheses. Target prediction highlighted a subset of genes showing convergent targeting by multiple independent blue-associated candidates, and duplex summaries showed structured, plant-like complementarity patterns (including frequent 10-11 pairing). Together, these results indicate that 3 h exposure to wavelength-defined light at 400 μmol m-2 s-1, particularly blue light, is associated with a distinct profile of detectable candidate small RNAs in Arabidopsis leaves and identify candidate interactions for follow-up validation.

5′ nucleotide bias↗

PubMed2006

A simple and convenient approach for isolating RNA from highly viscous plant tissue rich in polysaccharides.

RNA isolation is a prerequisite to the study of gene expression of herbaceous plant Dendrobium nobile under an environmental stress. However, RNA isolation is difficult in the plant of genus Dendrobium, which is relatively viscous, due to being rich in polysaccharides. The common protocols for RNA isolation are tedious and usually result in poor yields when applied to D. nobile. Here, we describe a simple and convenient method for high quality total RNA extraction from D. nobile. Main procedures are as follows: smashing plant tissue and cell wall by means of pre-cooled citrate homogenization, cleaving cell membrane by using guanidine hydrochloride lysis buffer, removing proteins, polyphenols and polysaccharides by acidic phenol/chloroform. It is particularly useful for processing large numbers of plant samples. The whole process can be completed within 2.5 h. The extracted RNA is suitable for applications such as RT-PCR, Northern analysis and screening of differential expression genes.

Cell Culture Techniques↗

PubMed2004

RNA degradation in human breast tissue after surgical removal: a time-course study.

There is much interest in the study of human malignancy using gene expression profiling techniques. Expression profiles obtained from microarrays utilize RNA extracted from the tissue in question. Currently, cell cultures or fresh tissue processed "quickly" are used in these studies. To our knowledge, there are no published reports of a time-course of RNA degradation in surgically removed breast tissue. Such a time-course study is critically needed. We obtained normal breast tissue from breast reduction surgery. Portions of breast tissue kept at room temperature were sampled and placed into RNAlater to preserve RNA at different time-points from 10 min to 3 h after the surgical removal. We evaluated total RNA integrity from each specimen using agarose gel electrophoresis and real-time quantitative RT-PCR analysis of four genes. Electrophoresis showed good-quality, intact RNA at all time points up to 3 h. Quantitative RT-PCR showed no difference in amplified products among all samples. Our study showed that there was no loss of RNA integrity in normal breast tissue for up to 3 h after surgical removal.

Actins↗

PubMed2005

The impact of RNA stabilization on minimal residual disease assessment in chronic myeloid leukemia.

BACKGROUND AND OBJECTIVES: Accurate quantification of BCR-ABL mRNA is of critical importance for managing patients with chronic myeloid leukemia (CML) who are receiving imatinib therapy. RNA degradation thus constitutes a potential problem for laboratories quantifying minimal residual disease (MRD). Patients' samples that take a long time to be transported from the hospital to the analyzing laboratory may be subject to RNA degradation with a corresponding loss in sensitivity and possible generation of false negative results. Recently, RNA preservation systems have been developed in order to improve RNA stability. The aim of the present study was to investigate such a system. DESIGN AND METHODS: We evaluated the performance of the PAXgene Blood RNA Kit in follow-up CML peripheral blood samples and compared the results to those from unstabilized parallel Trizol extracted samples. The different sample processing methods were evaluated by real-time polymerase chain reaction (PCR) analysis. RESULTS: RNA isolated with the PAXgene system gave a superior yield per milliliter of blood than did the routine Trizol extraction method. However, although of comparable quality, the RNA did not PCR-amplify as efficiently as equal amounts of RNA from routinely processed samples. Therefore, RNA processed with the PAXgene system showed decreased sensitivity for MRD detection, resulting in false negative results. The sensitivity was comparable to that of samples processed routinely 20-30 hours after phlebotomy. INTERPRETATION AND CONCLUSIONS: We conclude that routinely processed, i.e. unstabilized, peripheral blood that reaches the laboratory and is processed within 30 hours is preferable for MRD detection. Optimal results were achieved with fresh samples processed within 5 hours with the Trizol method. However, RNA stabilization may be useful if sample transit is expected to exceed 30 hours.

Gene Expression Regulation↗

PubMed1992

p53 gene expression in medulloblastoma by quantitative polymerase chain reaction.

The frequent cytogenetic abnormality--isochromosome 17q [i(17)q]--observed in medulloblastomas (MB) may result in altered expression of the oncosuppressor gene p53 that is located on 17p. p53 expression was therefore evaluated in five MBs and in one MB cell line derived from one of these tumors. Expression levels of p53 utilizing serially diluted unfractionated RNA from tumors and the cell line were assessed both by dot-blot and by reverse transcription (RT) followed by the polymerase chain reaction (PCR). The quality of RNA, efficiency of reaction, and transcript quantitation were determined by simultaneous transcription and amplification of a similarly sized fragment of the alpha-tubulin gene. All MBs showed low levels of expression of p53 compared to those found in normal tissues. p53 messenger RNA (mRNA) was significantly increased (two- to threefold) in the MB cell line compared to its tumor of origin and to the other MBs. Immunoperoxidase studies performed with monoclonal antibodies to the p53 protein product showed focal nuclear expression in one of five of the original tumors while most cells grown in vitro and in the xenograft were positive.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2002

Rapid isolation of cancer cells from tumor tissue by micromanuiplator and extraction of tiny amount of RNA.

OBJECTIVE: To establish a rapid method for isolating and purifying cancer cells from tumor tissue and for RNA extraction from tiny amount of the cells thus obtained. METHODS: Frozen sections of the tumor tissues were prepared followed by rapid staining. Clusters of the cancer cells were isolated from the sections by micromanipulation technique and purified for extracting intact RNA that was subsequently assayed. RESULTS: Clear vision was achieved by the staining of the sections. The cancer cell clusters were precisely isolated from which high-quality intact RNA was obtained as demonstrated by reverse transcriptase-PCR. CONCLUSION: Micromanuiplation can be effectively used in stead of laser capture microdissection to isolate and purify targeted cells from tiny amount of tissue samples, therefore making RNA extraction possible in this context.

Electrophoresis, Agar Gel↗

PubMed2002

A reverse transcription comparative real-time PCR method for quantitative detection of angiogenic growth factors in head and neck cancer patients.

OBJECTIVES: Head and neck cancer is one of the ten most frequent cancers in the world. The angiogenic growth factors VEGF, PDGF and bFGF play a role in cancer aggressiveness. We developed a sensitive method to quantify the gene expression of these factors in the tissues of head and neck cancer patients. DESIGN AND METHODS: All assays were performed using real-time RT-PCR, which yields a value (Ct) denoting the threshold cycle of PCR amplification at which product is first detected by fluorescence. The Ct is dependent on the quantity of the target molecule in the sample. To control for variation in RNA quantity and quality, we used 18S ribosome RNA as an internal control to calculate a relative Ct for the target molecules of interest, VEGF, PDGF and bFGF. A serially diluted positive control sample was analyzed by linear regression to determine the sensitivity and linearity of the assay. Paired normal and cancerous tissue samples from 115 head and neck cancer patients were assayed to ascertain the relative levels of the growth factors. RESULTS: The CVs of within-run and between-run assays for VEGF, PDGF and bFGF were all less than 3%. The correlation coefficient of the RNA concentrations and Ct values were 0.9987, 0.9977, and 0.9996 respectively for VEGF, PDGF and bFGF. The assay was sensitive to as little as 10(-3) ng of RNA. All three growth factors were significantly increased in tumor tissue as compared to normal tissue. VEGF, PDGF and bFGF levels were elevated in 71.3%, 58.2% and 54.0% of cancerous tissue samples, with average levels of over-expression of 35.1, 24.6 and 13. sixfold, respectively. CONCLUSION: This method provides sensitive, quantitative, high-throughput analysis for direct comparison of gene expression levels between samples, while adjusting for factors that may influence quantity determination. It should be applicable to molecules other than angiogenic growth factors, as well.

Endothelial Growth Factors↗