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PubMed2003

Moderate degradation does not preclude microarray analysis of small amounts of RNA.

Gene expression analysis by microarrays using small amounts of RNA is becoming more and more popular against the background of advances and increasing importance of small-sample acquisition methods like laser microdissection techniques. The quality of RNA preparations from such samples constitutes a frequent issue in this context. The aim of this study was to assess the impact of different extents of RNA degradation on the expression profile of the samples. We induced RNA degradation in human tumor and healthy tissue samples by endogeneous ribonucleases. Next, we amplified 20 ng total RNA degraded to different extents by two rounds of in vitro transcription and analyzed them using Affymetrix oligonucleotide microarrays. Expression differences for some genes were independently confirmed by real-time quantitative PCR. Our results suggest that gene expression profiles obtained from partially degraded RNA samples with still visible ribosomal bands exhibit a high degree of similarity compared to intact samples and that RNA samples of suboptimal quality might therefore still lead to meaningful results if used carefully.

Carcinoma, Renal Cell↗
PubMed2005

Optimization of liver biopsy RNA sampling and use of reference RNA for cDNA microarray analysis.

In this study, we used the rat liver as a model system to optimize the conditions for extracting RNA from liver biopsies for use in cDNA microarrays. We found that a 5-mm biopsy with a 16-gauge needle and storage in RNA later at 4 degrees C were optimal conditions for RNA extraction. The most important factor for the quantity and quality of RNA extraction was the sample diameter. Using the optimized sampling conditions and a cDNA microarray, we compared the expression of genes in the normal and the fibrotic tissues of the LEC rat liver, a model of liver tumorigenesis, with SD rat liver RNA as a reference. We found 29 genes that were up-regulated and 33 genes that were down-regulated in the fibrotic part of the liver. Furthermore, with the help of the reference RNA, we were able to classify the expression profiles into five groups without complex mathematical analyses; without the reference RNA, the genes could be classified into only two groups. Finally, we found that osteopontin was expressed at a very high level in the fibrotic portion of the LEC rat liver. This cDNA microarray result was validated by immunohistochemistry, which showed an elevated expression of osteopontin in the region of cholangiocarcinoma and a lack of expression in normal tissues. With optimized conditions, we should be able to apply the microarray system for routine practice.

Animals↗
PubMed2007

Real-time RT-PCR (TaqMan) assays for the detection of Grapevine Leafroll associated viruses 1-5 and 9.

Real-time RT-PCR (TaqMan) assays were developed for the specific detection of Grapevine Leafroll associated viruses 1-5 and 9 (GLRaV-1-5 and -9). The assays were evaluated against a wide range of geographically distributed isolates. Geographical locations included South Africa, Europe, Australia, Asia, Latin America and the United States. Sequences were piled up from the most conserved regions of these geographically diverse isolates and TaqMan primers and probes were designed, targeting the regions with 100% sequence identity. Improving the RNA extraction technique and testing the quality of the RNA using the 18S ribosomal RNA TaqMan assay as an RNA specific internal control to validate the quality of the extracted RNA proved to generate better diagnostic assays. The real-time TaqMan RT-PCR assays were compared to the conventional RT-PCR assays for the detection of viruses using purified total RNA as well as crude extract. The data showed that when using total RNA extracted either by the Qiagen RNeasy method or by an ABI automated system more isolates were detected in comparison to crude extract. The optimum volume of crude extract prepared in GES for use in real-time TaqMan RT-PCR cocktail was determined to be 1 microl per reaction. In addition this report showed that TaqMan RT-PCR was more sensitive than conventional one-step RT-PCR for testing different isolates of these viruses either using RNA or crude tissue extract.

Closteroviridae↗
PubMed2002

Production of bulk amounts of universal RNA for DNA microarrays.

In DNA microarray technology, repeatability and reliability are very important to compare multiple RNA samplesfrom different experiments. The application of common or universal RNA as a standard control equalizes the differences in hybridization parameters and array variations. For this purpose, high-quality reference RNA is necessary in bulk amounts. A novel approach was developed to get milligrams of sense or antisense RNA, starting from micrograms of pooled total RNA from different cell lines, tissues, or organisms. This method is inexpensive and allows further labeling procedures using poly(dT) or random oligomers as primers. In addition, amplified, sense reference RNA is suitable for standard labeling protocols, while the antisense reference RNA can be used with antisense RNA from the linear sample amplification method. Here we produced universal RNA for human, rat, and alfalfa and demonstrated the quality using specific cDNA microarrays.

Animals↗
PubMed2000

Assessment of template quality by the incorporation of an internal control into a RT-PCR for the detection of rabies and rabies-related viruses.

A method is described to assess RNA template quality by the incorporation of a ribosomal RNA (rRNA) internal (in tube) control into a standard rabies and rabies-related virus specific RT-PCR. Specific virus and rRNA templates were co-amplified in a duplex reaction from RNA extracts derived from 60 isolates representing all six of the established lyssavirus genotypes. To ensure a wide species applicability of this technique we demonstrated that the rRNA assay was capable of functioning using the cells or tissues of 14 different mammals. Parallel studies between the duplex and the unlinked lyssavirus assay demonstrated only a minor reduction in the sensitivity of the former test. The ribosomal and viral targets (unlike beta-actin RNA) were shown to have similar degradation kinetics making rRNA amplification a good control for viral target integrity. As a consequence, the use of this system would reduce the likelihood of obtaining false negative RT-PCR results from lyssavirus infected material.

Animals↗
PubMed2006

RNA extraction.

RNA extraction is a routine technique in a molecular biology lab. High quality of RNA extracted from plants is a prerequisite to succeeding in subsequent experiments such as reverse transcription-polymerase chain reaction, Northern hybridization, cDNA library construction, and microarray analysis. Two methods for RNA extraction in Arabidopsis, small-scale and large-scale, are described here. RNA isolated by using the small-scale method can be applied in most downstream applications. The large-scale RNA preparation is recommended to extract RNA for Northern hybridization only.

Arabidopsis↗
PubMedDate not supplied

Expression of the 3-hydroxy-3-methylglutaryl coenzyme A-reductase and LDL-receptor genes in human embryonic tumours and in normal foetal tissues.

The expression of the HMG-CoA-reductase and LDL-receptor genes was examined in primary human embryonic tumours and in normal human first trimester foetal tissues. All embryonic and extra-embryonic tissues examined contained RNA that hybridized with the HMG-CoA-reductase and LDL-receptor cDNA-probes. However, the transcript levels varied considerably between organs. By using Northern blotting, we confirmed that normal foetal tissues contained HMG-CoA-reductase and LDL-receptor transcripts of normal size (4.2 kB and 5.3 kB, respectively). All human developmental tumours examined were characterized by high levels of HMG-Co-A-reductase RNA. However, when the quality of this RNA was examined in greater detail it was found that the 4.2 kB band only accounted for a minor portion of the RNA. Instead we found a range of other transcripts (3.1, 1.1., 0.95, 0.78 and 0.55 kB) that hybridized with the probe. There was a qualitative difference in that only the non-seminomas contained high levels of the three smaller transcripts (0.95, 0.78 and 0.55 kB). In contrast, only one tumor--an embryonal carcinoma--contained increased levels of LDL-receptor transcript. This was found to be of the expected size (5.3 kB).

Cell Division↗
PubMed2026

Optimized Hot Phenol-Based RNA Extraction from Mycobacteria: A Robust Approach for Reliable Gene Expression Analysis.

Mycobacterium tuberculosis (Mtb) remains a major global health threat, underscoring the need for reliable transcriptomic studies to understand its biology and drug resistance mechanisms. Such analyses depend on obtaining high-quality, high-yield RNA. Although several RNA extraction methods are available, many require expensive reagents, large culture volumes, or specialized equipment, limiting their suitability for large-scale studies, particularly in resource-constrained settings. Here, an optimized Hot Phenol based RNA extraction method specifically tailored for mycobacteria is presented. The method uses minimal culture volume and commonly available reagents to consistently yield high-quality RNA suitable for high-throughput transcriptomic applications. RNA quantity and integrity were assessed by gel electrophoresis and RNA integrity analysis (RIN), and its suitability for downstream applications was confirmed by qPCR and Qubit 4. To benchmark the performance of the optimized method, a parallel RNA extraction using TRIzol and RNeasy under identical experimental conditions was carried out, including the same Mycobacterium species, culture volume, growth phase (logarithmic and stationary), and lysis conditions. This allowed a direct comparison of yield, quality, feasibility, and cost. The optimized Hot Phenol method demonstrated comparable or improved RNA yield and quality while significantly reducing reagent cost and dependence on specialized equipment. Owing to its efficiency, reproducibility, and affordability, this protocol provides a practical alternative for large-scale gene expression and transcriptomic studies in Mtb and other mycobacterial species.

RNA, Bacterial↗
PubMed2004

Genetics of dizygotic twinning: a feasibility study for a biobank.

To locate the genes that make a substantial contribution to variation in natural dizygotic twinning in humans, large-scale studies are needed. New studies should not stop at DNA genotyping, but collect material that allow gene-expression analysis, transcriptomics, proteomics and endocrinology. In this article we describe a pilot study to examine the feasibility, effectiveness and logistics of large-scale nationwide sample collection in Dutch families in which two or more sisters have given birth to spontaneous dizygotic twins. Pedigree data and addresses from family members of proband mothers were collected by telephone. Blood and urine samples were collected during a home visit, and handled in the afternoon. All participants were bled between 7 a.m. and 10 a.m. after overnight fasting. Blood samples of fertile women with a natural cycle were collected on the second, third or fourth day of their menstrual cycle. The effects of transportation and storage on blood quality, lipids, RNA with and without challenge, lymphocytes and other parameters were examined. Genomic DNA was isolated from blood and cells were immortalized using Epstein-Barr virus. In 78.6% of the women with a natural cycle blood samples were collected on the second, third or fourth day of the menstrual cycle. This percentage is likely to increase with the more dense geographical distribution of participants in the larger population. We conclude that the pilot study demonstrated the feasibility of this protocol to collect good quality of plasma, DNA, RNA and lymphocyte samples by home visits.

DNA↗
PubMed2005

Simpleaffy: a BioConductor package for Affymetrix Quality Control and data analysis.

UNLABELLED: Quality Control is a fundamental aspect of successful microarray data analysis. Simpleaffy is a BioConductor package that provides access to a variety of QC metrics for assessing the quality of RNA samples and of the intermediate stages of sample preparation and hybridization. Simpleaffy also offers fast implementations of popular algorithms for generating expression summaries and detection calls. AVAILABILITY: Simpleaffy can be downloaded from http://www.bioconductor.org. SUPPLEMENTARY INFORMATION: Additional information can be found on the supplementary website located at http://bioinformatics.picr.man.ac.uk.

Computational Biology↗
PubMed2002

Improvement of molecular monitoring of residual disease in leukemias by bedside RNA stabilization.

The sensitivity of assays designed to monitor minimal residual disease (MRD) by RT-PCR in leukemia depend on quality and quantity of RNA derived from peripheral blood (PB) and bone marrow (BM) leukocytes. Shipment of material may lead to RNA degradation resulting in a loss of sensitivity and, potentially, false negative results. Furthermore, degradation may lead to inaccurate estimates of MRD in positive specimens. We sought to determine feasibility and efficacy of a novel blood collection and processing system which is based on integrated RNA stabilization at the time of phlebotomy (PAXgene Blood RNA Kit) by comparison with standard methods of RNA extraction (cesium chloride gradient ultracentrifugation and RNeasy Mini Kit) using unstabilized EDTA anticoagulated PB. In 26 patients with chronic myelogenous leukemia (CML) on therapy, PB was processed after a storage time at room temperature of 2 and 72 h according to these protocols. BCR-ABL, total ABL and glucose-6-phosphate dehydrogenase (G6PD) mRNA transcripts of PB samples were quantified as a measure for response to therapy and RNA integrity. RNA yield expressed as the ratio of ABL transcripts after a storage time of 72 h/ABL transcripts after a storage time of 2 h at room temperature was significantly higher with the stabilizing method (median 0.40) compared to the RNeasy method using unstabilized PB (median 0.13, P = 0.01). Furthermore, ratios BCR-ABL/ABL after 72 vs 2 h still correlated well using the PAXgene method (r = 0.99, P < 0.0001) in contrast to the standard method which did not (r = 0.65, P = 0.03). Even investigation of complete cytogenetic responders with very low tumor burden showed a good correlation of ratios BCR-ABL/ABL compared to the reference method. Comparable results were achieved using G6PD transcripts as standard. We conclude that the new PAXgene stabilization method could improve RNA quality and the comparability of molecular monitoring within and between multicenter trials.

Adult↗
PubMed2001

Quantitative determination of gene expression in human lymphocytes assessed by reverse transcription-polymerase chain reaction coupled to high-performance liquid chromatography.

Gene expression in human lymphocytes was assessed using reverse transcription and polymerase chain reaction amplification followed by ion-pair reversed-phase chromatography analysis. Competitive PCR was used to quantitate the desired cDNAs with a polivalent competitor adaptable to multiple novel mRNAs estimations with minor changes. Accuracy was 11.27+/- 11.87% (n = 7), as determined using standards. The coefficients of variation of the assessment of human OK12b were 7% (n = 6), 7.68 attmol/microg of total RNA, and 21% (n = 6), 0.93 attmol/microg of total RNA. Sample-to-sample variation in the reverse transcription and in the quantity and quality of RNA was attenuated by normalising results to beta-actin mRNA expression. The correlation between the OK12b/beta-actin ratio and competitive assessments of OK12b was 0.984, n = 6. The correlation between HPLC results and an independent method based on radionuclide uptake by the product, detected by electrophoretic separation, was 0.848, n = 10.

Base Sequence↗
PubMed2006

Laser capture microdissection of plant cells from tape-transferred paraffin sections promotes recovery of structurally intact RNA for global gene profiling.

Laser capture microdissection and related technologies permit the harvest of individual cells and cell types. Isolation of either nucleic acids or proteins from laser-captured cells supports such downstream applications as the construction of cell-specific cDNA libraries and the profiling of expressed genes and proteins. The success of these endeavors is dependent upon the yield, purity and structural integrity of the macromolecules derived from harvested cells. Here, we report protocols that promote the isolation of structurally intact RNA from laser-captured cells of paraffin-embedded tissues. The use of a tape transfer system that obviates the need to wet paraffin sections prior to slide mounting significantly increases RNA structural quality. Integrity is assessed directly via electrophoretic separation of picogram-nanogram levels of total RNA isolated from multiple cell types, including those comprising Arabidopsis ovules, replums and stamen abscission zones. RNA prepared from specialized cells within siliques provided targets for profiling the Arabidopsis genome during replum cell development. Digital northern analysis of transcripts expressed near the threshold of the system's ability to score signal presence suggests that low-abundance transcripts representing as little as approximately 0.002% of total mRNA can be reliably detected. Microarray data reveal a significant shift from primary cell-wall metabolism to lignin biosynthesis in replum tissues during fruit maturation.

Arabidopsis↗
PubMed2006

[Construction of the female subtractive cDNA library and screening of the specific expressing genes].

OBJECTIVE: To screen the Schistosoma japonicum female specific expressing genes. METHODS: S. japonicum adult worms were collected from the rabbits' vein after six-week infection by affusing method. The adult worms were stabilized by RNA-later liquid, the male and female worms were carefully separated with nipper. The high quality total RNA was extracted and mRNA was obtained after purification. Double stranded cDNAs were synthesized after reverse transcription. Female subtractive (female as tester, male as driver) and male subtractive (male as tester, female as driver) cDNA libraries were constructed. The differentially expressed genes were further screened by dot-blot hybridization. The clones were selected and sequenced, which showed apparently higher signals when hybridizing with the female subtracting male probes, than those signals when hybridizing with the male subtracting female probes. The homology of these sequences was searched with BLAST program. The semi-quantitative PCR was applied to test the differential gene expression in female and male adult worms. RESULT: Female subtracting male and male subtracting female cDNA libraries were constructed with SSH technique. After dot-blot hybridization, 50 clones were tested to be the potential female differentially expressed genes and were sequenced. 42 expressing sequence tags (ESTs) were received. After bioinformatics analysis, 17 fragments (about 40.5%) showed high identity with the S. japonicum egg-shell protein genes, 17 sequences (about 40.5%) were highly homologous to unknown S. japonicum genes and partly homologous to female specific 800 protein. 8 fragments (about 19.0%) showed high identity with other S. japonicum unknown genes. The fragments in clones of 577, 579, 668, 695, 720, and 708 were tested by RT-PCR to be the differentially expressed genes in female adult worms using S. japonicum actin gene as the internal standard. These fragments were highly homologous to S. japonicum egg shell protein gene AY222885, AY222895, AB017097, AF519182, M32281, and S. japonicum unknown gene AY813556 respectively. CONCLUSION: SSH is essential to screen the differentially expressed genes of S. japonicum female worms. A number of female specific genes have been found by this method.

Animals↗
PubMed2005

Assessment of fixatives, fixation, and tissue processing on morphology and RNA integrity.

Molecular characterization of morphologic change requires exquisite tissue morphology and RNA preservation; however, traditional fixatives usually result in fragmented RNA. To optimize molecular analyses on fixed tissues, we assessed morphologic and RNA integrity in rat liver when sections were fixed in 70% neutral-buffered formalin, modified Davidson's II, 70% ethanol, UMFIX, modified Carnoy's, modified methacarn, Bouin's, phosphate-buffered saline, or 30% sucrose. Each sample was subjected to standard or microwave fixation and standard or microwave processing, and sections were evaluated microscopically. RNA was extracted and assessed for preservation of quality and quantity. Modified methacarn, 70% ethanol, and modified Carnoy's solution each resulted in tissue morphology representing a reasonable alternative to formalin. Modified methacarn and UMFIX best preserved RNA quality. Neither microwave fixation nor processing affected RNA integrity relative to standard methods, although morphology was modestly improved. We conclude that modified methacarn, 70% ethanol, and modified Carnoy's solution provided acceptable preservation of tissue morphology and RNA quality using both standard and microwave fixation and processing methods. Of these three fixatives, modified methacarn provided the best results and can be considered a fixative of choice where tissue morphology and RNA integrity are being assessed in the same specimens.

Animals↗
PubMed1992

Expression of low density lipoprotein receptor, apolipoprotein AI, AII and AIV in various rat organs utilizing an efficient and rapid method for RNA isolation.

Intact RNA from various rat organs was isolated by an efficient and rapid method. This method of RNA isolation is a modification of an earlier method that uses guanidinium isothiocynate followed by extraction in the presence of sarcosyl, acetate and phenol. The RNA obtained by the method reported here was comparable with the RNA prepared by the CsCl2 ultracentrifugation method and the commercially available kit based on published methods. The quality of RNA was found suitable for Northern blotting analysis, RNase protection assays and reverse transcriptase-polymerase chain reaction (RT-PCR). Since reverse transcriptase is active in the buffer used for Taq DNA polymerase, only one reaction needs to be set up. We also found that the use of aurintricarboxylic acid in the RNA preparation prevents the degradation of RNA during storage. Expression of low density lipoprotein (LDL) receptor, apolipoprotein (apo) AI, AII and AIV mRNAs were quantified in various rat organs. Our results indicated that rat LDL receptor mRNA is expressed in several organs whereas apoAI and AIV mRNAs were expressed mainly in the liver and intestine. However, apo AII mRNA is expressed mainly in the liver. Unlike mice and some species of monkeys, in the rat apoAI mRNA is expressed at 5-6 times higher levels in the intestine compared to liver. Apo AIV mRNA abundance was also found to be several fold higher in intestine compared to hepatic tissues. We present here, for the first time, data on the absolute amounts of LDL receptor, apoAI, AII and AIV mRNA in various rat organs which were quantified by a novel RNase protection/solution hybridization assay.

Animals↗
PubMed2002

Optimizing gene expression analysis in archival brain tissue.

Analysis of gene expression in the brain is a valuable tool to study the function of the brain under normal and pathological conditions. Although there are many techniques used to measure gene expression the validity of any such experiment is directly related to the quality of the RNA in the samples. The most readily available source of human brain tissue is post-mortem and while frozen tissue is sometimes available, most archived tissue is fixed and paraffin-embedded. The use of fixed tissue for expression analysis introduces variables, which must be considered in the experimental design. In addition, factors associated with clinical variability of the patient and with tissue procurement can affect RNA transcript levels. In order to illustrate the effects of two common tissue fixatives, formalin and ethanol, on the quality of RNA for expression analysis we compare RNA extracted from these fixed tissues to the gold standard, flash-frozen tissue. We describe RNA extraction from fixed tissue and ways to assess the quality or intactness of the RNA using reverse transcription combined with polymerase chain reaction amplification. An advantage of using archived tissue is the ease with which single cells or subpopulations of cells can be obtained by laser microdissection. The successful isolation of RNA from microdissected cells is also presented. From our results and a review of the literature we conclude that RNA from fixed tissues is a viable source of RNA for expression analysis which should enable new experimental approaches and discoveries as long as attention is given to variables that can affect RNA at all levels of analysis.

Archives↗