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The Ro 60 kDa autoantigen binds misfolded RNAs and likely functions in small RNA quality control. In this issue of Cell, Stein et al. (2005) present crystal structures of Ro alone and bound to both double- and single-stranded RNA, revealing two distinct RNA binding sites that suggest how Ro may distinguish between native and misfolded small RNAs.
Total RNA was extracted from 105 individual postmortem human brain samples representing a range of postmortem conditions. To improve upon parameters currently used to screen for RNA quality, electropherogram patterns generated by the Agilent Bioanalyzer 2100 were compared to the average score in random hexamer-primed reverse transcription real-time PCR for four housekeeping genes in each RNA sample. The ribosomal ratio (28S to 18S) was found to be unrelated to the housekeeping gene score (r = -0.06; P = 0.50), and there was no threshold value in the ratio that could be applied to effectively categorize the RNA degradation. Although the housekeeping gene score correlated significantly with the percentage of area in the electropherogram corresponding to moderate to high molecular weight intact mRNA (r = 0.41; P = 0.0001), the best discriminator was determined to be the ratio of the 18S peak height to the highest peak in the tRNA to 18S rRNA baseline. Applying a lower boundary of 2.12 for the ratio allowed for the screening out of samples with the lowest housekeeping gene scores without excluding better-quality samples. This measure represents a marked improvement over the 28S to 18S ratio, which proved to be a misleading indicator of the state of the mRNA for use in random hexamer-primed reverse transcription PCR.
An efficient method for the isolation of RNA from cartilage is described. The difficulties in obtaining RNA from cartilage, a tissue of low cell density and high proteoglycan content, were overcome by making several modifications to the guanidine thiocyanate/cesium chloride method of RNA extraction. Cartilage tissue is frozen, crushed, and homogenized in a 4 M guanidine thiocyanate lysis buffer. The RNA is then pelleted by ultracentrifugation through a cesium trifluoroacetate density gradient. The use of cesium trifluoroacetate, rather than cesium chloride, for density gradient centrifugation improves both the yield and purity of total RNA isolated from cartilage. The ultracentrifugation has been adapted to the Beckman TL100 tabletop centrifuge and is complete in 3 h. This fast, simple method produces high quality RNA, suitable for use in RNase protection assays, polymerase chain reaction analysis, and Northern analysis. This purification procedure may be applicable to other sources, from which RNA isolation is complicated by the presence of abundant cell wall or matrix components.
Optimal cutting temperature (OCT) is a widely used embedding medium for tissues for histopathologic analysis. This investigation examined the effects that OCT storage can have upon the ability to perform subsequent molecular biological analyses. Tumor material was dissected into small pieces and stored at approximately 20 degrees C both with and without OCT. DNA and RNA were then extracted from the tissue fragments and analyzed by the polymerase chain reaction (PCR), using primer sets designed to amplify a range of product sizes, and also by reverse transcriptase-PCR (RT-PCR). The storage of pathological specimens in OCT compound was found to affect significantly and irreversibly the ability to amplify DNA in the PCR, particularly as the size of the amplified fragment increased. This effect appeared to occur as a result of greater degradation of DNA extracted from tissue embedded in OCT compared to DNA extracted from tissue stored without OCT. RNA quality appeared unaffected, which may be because of the extraction protocol employed. Our results suggest that OCT-embedded frozen-tissue samples may be used for RNA isolation for subsequent RT-PCR and for the in vitro amplification of DNA targets of approximately < 300 base pairs only. We strongly advise against the routine storage of any tissue biopsy material in OCT if molecular analyses may be required.
RNA isolation from yeast is complicated by the need to initially break the cell wall. While this can be accomplished by glass bead disruption or enzyme treatment, these approaches result in DNA contamination and/or the need for incubation periods. We have developed a protocol for the isolation of RNA samples from yeast that minimizes degradation by RNases and incorporates two purification steps: acid phenol extraction and binding to a silica matrix. The procedure requires no precipitation steps, facilitating automation, and can be completed in less than 90 min. The RNA quality is ideal for microarray analysis.
BACKGROUND: The applicability of microarray-based transcriptome massive analysis is often limited by the need for large amounts of high-quality RNA. RNA arbitrarily primed PCR (RAP-PCR) is an unbiased fingerprinting PCR technique that reduces both the amount of initial material needed and the complexity of the transcriptome. The aim of this study was to evaluate the feasibility of using hybridization of RAP-PCR products as transcriptome representations to analyze differential gene expression in a microarray platform. METHODS: RAP-PCR products obtained from samples with limited availability of biological material, such as experimental metastases, were hybridized to conventional cDNA microarrays. We performed replicates of self-self hybridizations of RAP-PCR products and mathematical modeling to assess reproducibility and sources of variation. RESULTS: Gene/slide interaction (47.3%) and the PCR reaction (33.8%) accounted for the majority of the variability. From these observations, we designed a protocol using two pools of three independent RAP-PCR reactions coming from two independent reverse transcription reactions hybridized in duplicate and evaluated them in the analyses of paired xenograft-metastases samples. Using this approach, we found that HER2 and MMP7 may be down-regulated during distal dissemination of colorectal tumors. CONCLUSION: RAP-PCR glass array hybridization can be used for transcriptome analysis of small samples.
The analysis of gene expression by growth plate chondrocytes in vivo has been hampered by the inherent difficulty in performing in situ hybridization on mineralized tissues. The combination of laser capture microdissection and reverse transcription-polymerase chain reaction (RT-PCR) allows analysis of gene expression by cells selectively removed from histologic sections by laser ablation. In order to apply this method to mineralized tissues, a decalcification process is required. The object of this study was to determine the optimal method for tissue decalcification prior to laser capture microdissection RT-PCR that will preserve integrity of the mRNA population. Acetone, 10% formalin, and methacarn were evaluated as fixatives, while Surgipath Decalicifier I, 10% ethylenediaminetetraacetic acid (EDTA), and 20% EDTA were evaluated as decalcifying reagents. Our results demonstrate that the optimal RNA quality was preserved by a decalcification protocol consisting of 20% EDTA for decalcification followed by fixation in methacarn, although this method is also associated with a reduction in RNA quantity.
OBJECTIVES: To examine vascular endothelial growth factor (VEGF), VEGF-receptor-(R)1, and R2 mRNA levels in renal cell carcinoma (RCC), a tumour generally refractory to most medical therapy, but for which a potentially useful therapeutic alternative is inhibition of angiogenesis. PATIENTS AND METHODS: VEGF, VEGF-R1 and -R2 mRNA levels were analysed using the quantitative reverse transcription-polymerase chain reaction. RNA was extracted from 84 conventional (clear cell) RCCs (cRCC), 20 papillary (pRCC), six chromophobe (chRCC), and 27 corresponding kidney cortex tissues, obtained from 110 patients in whom high-quality RNA was available from the tumours (53 women and 57 men, mean age 64.7 years, range 25-85). RESULTS: The VEGF, VEGF-R1, and -R2 mRNA levels were higher in tumour than in kidney cortex tissues. Among the RCC types, cRCC had higher VEGF levels than pRCC. In cRCC, VEGF-R2 levels were higher in stage I-II than in more advanced stages. In pRCC, VEGF and VEGF-R2 levels were higher in stage III than in stage I-II tumours. In cRCC, patients with VEGF levels below the median had a significantly shorter survival time than those with higher levels. By contrast, in pRCC, VEGF, VEGF-R1 and -R2 RNA levels above the median were related to adverse survival. Using multivariate analysis in cRCCs, VEGF-R1 mRNA level was the last factor to be omitted after stepwise elimination analysis. CONCLUSION: VEGF and its receptors were associated with tumour stage and survival, but were not independent prognostic factors. Different RCC types had different expression patterns of VEGF and receptor mRNA levels. We conclude that different pathways might be involved in regulating angiogenesis in the specific RCC types. Detailed knowledge of angiogenesis in RCC is essential when designing new treatment trials where angiogenesis inhibition is used.
Expression-profiling studies have helped define genetic changes associated with carcinogenesis. Determining which alterations in gene expression are causally associated with cancer and which result from the general dysregulation in gene expression that is characteristic of malignancies remains a problem. Transcriptional profiling of early lesions (small cancers or precancers) holds promise for identifying biologically important changes in gene expression. There are, however, technical barriers to the study of small tumors. The total number of cells available for analysis is limiting. It is also often difficult to distinguish cancer cells from normal proliferating cells in frozen sections that are typically used as a source of RNA. Here we describe an ethanol fixation and paraffin-embedding protocol that preserves tissue architecture and cellular morphology of the mouse endometrium, and allows for the recovery of high-quality RNA from microdissected cells. We performed GeneChip expression profiling using RNA from 800 to 4400 cells microdissected from ethanol-fixed, paraffin-embedded uteri. Endometrial adenocarcinomas exhibited changes in the levels of a number of messages known to be abnormally expressed in cancer, and differential expression of additional transcripts not previously implicated in carcinogenesis. We confirmed increased Amd1 expression in RNAs from mouse endometrial carcinomas that were hybridized to GeneChips and validated overexpression of this transcript in additional tumors.
In previous studies, we detected a frame shift mutation in the gene encoding the autoantigen La of a patient with systemic lupus erythematosus. The mutant La mRNA contains a premature termination codon. mRNAs that prematurely terminate translation should be eliminated by RNA quality control mechanisms. As we find Abs specific for the mutant La form in approximately 30% of sera from anti-La-positive patients, we expected that mutant La mRNAs circumvent RNA control and the expression of mutant La protein could become harmful. Indeed, real-time PCR, immunostaining, and immunoblotting data of mice transgenic for the mutant La form show that mutant La mRNAs are not repressed in these animals and are translated to mutant La protein. In addition to the mutant La protein, we detected a minor portion of native human La in the mutant La-transgenic mice. Therefore, ribosomal frame shifting may allow the mutant La mRNA to escape from RNA control. Interestingly, expression of the mutant La mRNA results in a lupus-like disease in the experimental mice. Consequently, escape of mutant La mRNA from RNA control can have two effects: it 1) results in the expression of an immunogenic (neo)epitope, and 2) predisposes to autoimmunity.
BACKGROUND: The TEL/AML1 fusion associated with t(12;21)(p13;q22) is the most common gene rearrangement in childhood malignancy, occurring in approximately 25% of pediatric acute lymphoblastic leukemia. The TEL/AML1 rearrangement is cryptic at the cytogenetic level but confers a favorable prognosis. The AML1 gene was first identified by virtue of its involvement in adult and pediatric acute myeloid malignancies associated with t(8;21) and t(3;21)(q26;q22.1). We have therefore determined the frequency of the TEL/AML1 fusion in pediatric myeloid leukemias by RT-PCR analysis. METHODS: Total RNA was isolated from cryopreserved bone marrow samples of 38 pediatric patients with AML. RNA quality was controlled for by amplification of the TEL gene. An RT-PCR assay was then used to test for the presence of the TEL/AML1 fusion. RESULTS: 29 patients had adequate RNA for analysis. Zero out of 29 pediatric AML patients had evidence for the TEL/AML1 fusion by RT-PCR. CONCLUSIONS: The TEL/AML1 fusion does not occur in children with AML and suggests that the TEL/AML1 rearrangement is restricted in pediatric hematologic malignancy to B lineage ALL.
We have developed a simple protocol for isolating RNA from both cell culture and tissue from human and animal sources, using guanidine thiocyanate and guanidine hydrochloride, but no organic solvents. The protocol reproducibly yielded 15 to 25 micrograms of high-quality RNA per 10(6) cells of human and animal origin and 1 to 1.1 mg of RNA per gram of human placental tissue. The RNA so obtained was ribonuclease-free and not contaminated by DNA. It was suitable for reverse transcription-polymerase chain reaction, Northern blot analysis, and in vitro expression of proteins. Thus, the molecular assessment of both research and clinical samples can be readily and reliably initiated by the application of this protocol.
Streptomycetes are complex soil bacteria capable of producing aerial reproductive mycelium and secondary metabolites. We observed novel phenomena such as an extended life cycle including flotation and anaerobiosis using standing liquid cultures. This paper describes an improved method for isolating good quality RNA from standing liquid cultures of S. coelicolor via excellent cell lysis.
Current methods of analytical RNA electrophoresis are based on the utilization of either complicated laboratory instrumentation or toxic, carcinogenic, or expensive chemicals. We suggest here the use of classical Tris-acetate-ethylenediamine tetraacetic acid (TAE) agarose gels combined with prior denaturation of RNA samples in hot formamide for the electrophoretic separation of RNA species. We present a brief comparison of the proposed TAE/formamide method with the most common 3-(N-morpholino)propanesulfonic acid/formaldehyde agarose gel protocol and show that both methods produce comparable results for size determination of RNA molecules and subsequent Northern blotting of gels. In addition to purified RNA samples, the robustness of the TAE/formamide protocol is demonstrated by its suitability for the analysis of RNA quality in crude yeast cell lysates containing large amounts of proteins, DNA, and other contaminating molecules. We therefore propose the TAE/formamide agarose electrophoresis as a rapid, simple, and cheaper alternative to current methods of RNA electrophoresis. Additionally, another benefit is the reduced exposure of laboratory personnel to hazardous chemicals.
Ubiquitous RNases are the usual causes of RNA degradation on its isolation from mammalian cells. Using guanidine hydrochloride for the extraction of RNA from mouse peritoneal macrophages, we identify a major source of RNA-degrading activity, the stage of the extraction procedure at which this activity may be detected and show that its removal early in the extraction leads to a more dependable method for the recovery of high quality RNA.
In the present study, we have investigated whether RNA can be efficiently isolated from Bouin-fixed or formalin-fixed, paraffin-embedded lymphoid tissue specimens. To this aim, we applied a new and simple method that includes the combination of proteinase K digestion and column purification. By this method, we demonstrated that the amplification of long fragments could be accomplished after a pre-heating step before cDNA synthesis associated with the use of enzymes that work at high temperature. By means of PCR using different primers for two examined genes (glyceraldehyde-3-phosphate dehydrogenase [GAPDH]- and CD40), we amplified segments of cDNA obtained by reverse transcription of the isolated RNA extracted from Bouin-fixed or formalin-fixed paraffin-embedded tissues. Amplified fragments of the expected sizes were obtained for both genes tested indicating that this method is suitable for the isolation of high-quality RNA. To explore the possibility for giving accurate real time quantitative RT-PCR results, cDNA obtained from matched frozen, Bouin-fixed and formalin-fixed neoplastic samples (two diffuse large cell lymphomas, one plasmacytoma) was tested for the following target genes: CD40, Aquaporin-3, BLIMP1, IRF4, Syndecan-1. Delta threshold cycle (DeltaC(T)) values for Bouin-fixed and formalin-fixed paraffin-embedded tissues and their correlation with those for frozen samples showed an extremely high correlation (r > 0.90) for all of the tested genes. These results show that the method of RNA extraction we propose is suitable for giving accurate real time quantitative RT-PCR results.
Arboviral diseases represent a major public health challenge, especially in tropical regions where environmental conditions may favor the proliferation and spread of mosquito vectors. Thus, early and accurate detection of chikungunya virus (CHIKV) in mosquito populations can be a valuable tool for effective surveillance of circulating variants and for identifying new viral introductions. Given the challenges of detecting arboviruses in field-captured mosquitoes, we describe an integrated workflow for CHIKV molecular detection and whole-genome sequencing. This protocol includes mosquito homogenization using a bead-based mechanical disruptor, RNA extraction using TRIzol reagent with minor modifications, molecular screening using CHIKV-specific RT-qPCR, and whole-genome amplification followed by sequencing on Illumina platforms. Despite the protocol being optimized for individual mosquitoes, it results in high-quality RNA suitable for both entomological surveillance and genomic analysis. As this protocol allows recovery of complete CHIKV genomes from mosquito specimens, it can serve as a basis for genomic epidemiology studies, enabling monitoring of viral diversity and lineage dynamics, and facilitating early detection of emerging variants to support timely and targeted public health interventions in endemic and at-risk regions.
Acute promyelocytic leukemia (APL) is a medical emergency which requires rapid diagnosis and tailored treatment. Detection of the PML/RARalpha fusion gene in APL blasts is critical to start promptly the specific therapy with all-trans retinoic acid (ATRA). APL lacking this genetic lesion have been reported as being ATRA resistant. Reverse-transcription polymerase chain reaction (RT-PCR) has been extensively used to detect the PML/RARalpha cDNA. The reported PML/RARalpha amplification techniques are laborious and time consuming, and include conventional RNA extraction, cDNA synthesis and a two-round (nested) PCR. We hereby describe a few variations of the commonly adopted RNA extraction and PML/RARalpha RT-PCR protocols which allow a molecular diagnosis of APL to be carried out in less than 5 h. Processing of small volumes of leukemic cell lysate (0.5 ml) in a microfuge allows extraction of good quality RNA in 1 h. After reverse transcription to obtain cDNA, a 'hot start' PCR procedure was adopted which enabled us to amplify clearly visible and specific products after a single (not nested) amplification round. The PML/RARalpha fusion gene was detected in the blasts of six consecutive APL at diagnosis, and an APL-tailored protocol including ATRA was started in each case within 6 h of admission. On repeated experiments, the assay proved highly specific and sensitive for the rapid detection of all PML/RARalpha transcript types. Our data should encourage the use of this rapid procedure for the diagnosis of both typical APL and, particularly, less typical cases awaiting urgent therapeutic intervention.