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PubMed2004

Effect of formalin on extraction of mRNA from a formalin-fixed sample: a basic investigation.

BACKGROUND: The validity of molecular studies using RNA extracted from decades-old formalin-fixed and embedded tissue blocks is well documented. Formalin-fixed samples are, however, known to be poor materials for molecular biological applications. The aim of this study was to investigate the effect of formalin as a preservation agent on RNA quality and relevant molecular applications. METHODS: The quantity and quality of RNA from liver tissues are modified by the fixation time. Rat liver tissues were harvested and fixed for 4, 8, 12, and 24 h in 10% buffer formalin, and only 9% of RNA could be extracted from the formalin-fixed tissue stored for 4 h. RESULTS: Agarose gel electrophoresis did not show the characteristic pattern of RNA with 18S and 28S in all lanes. Based on the results, it is thought that the RNA was damaged owing to one or more of the following reasons: 1) formalin directly degrades RNA, 2) formalin inactivates RNase, and/or 3) formalin destroys the reagent for extraction of RNA. In this study, it was shown by electrophoresis that very little RNA was extracted by Trizol with 1% formalin. CONCLUSIONS: Formalin has direct and indirect ill effects on RNA during the extraction process.

Animals↗
PubMed2005

Assessing quality of hybridized RNA in Affymetrix GeneChip experiments using mixed-effects models.

The technology for hybridizing archived tissue specimens and the use of laser-capture microdissection for selecting cell populations for RNA extraction have increased over the past few years. Both these methods contribute to RNA degradation. Therefore, quality assessments of RNA hybridized to microarrays are becoming increasingly more important. Existing methods for estimating the quality of RNA hybridized to a GeneChip, from resulting microarray data, suffer from subjectivity and lack of estimates of variability. In this article, a method for assessing RNA quality for a hybridized array which overcomes these drawbacks is proposed. The effectiveness of the proposed method is demonstrated by the application of the method to two microarray data sets for which external verification of RNA quality is known.

Biometry↗
PubMed2001

Efficient isolation of total RNA from Clostridium without DNA contamination.

Several molecular techniques require high-quality RNA, free from DNA. Various methods have been described to obtain RNA to be used in expression studies or as starting material in differential display-reverse transcriptase (dd-RT-PCR), for which high-quality RNA free from DNA is an essential requirement. In this report, we compare three different methods to isolate RNA from Gram-positive bacteria: (1) An acid-phenol extraction protocol. (2) The "RNeasy mini kit" from QIAGEN (Valencia, CA, USA). (3) The "SV Total RNA Isolation System" from Promega (Madison, WI, USA).The QIAGEN-kit delivers the highest amount of RNA with the highest purity. Slot blot analysis and dd-RT-PCR confirm the absence of DNA contamination and Northern blot analysis and dd-RT-PCR show high quality of the extracted RNA. This RNA extraction method thus addresses current problems by permitting rapid and safe isolation with high yields of intact RNA for subsequent analysis.

Blotting, Northern↗
PubMed2001

Optimal procedure for extracting RNA from human ocular tissues and expression profiling of the congenital glaucoma gene FOXC1 using quantitative RT-PCR.

PURPOSE: To develop methods for obtaining high quality RNA from human donor eyes and to determine the expression profile of the congenital glaucoma gene FOXC1 in human ocular tissues. METHODS: To obtain high quality RNA from donor eyes, several different preservation methods were tested including storing eyes on ice, freezing eyes, and placing eyes in the commercial fixative RNAlaterTM prior to dissection and RNA extraction. Nine different ocular tissues from human donors were dissected and examined. Pigment-free total RNA was isolated and used for quantitative real-time RT-PCR using FOXC1 and GAPDH (internal standard) primers to assess the quality and expression of FOXC1. RESULTS: An expression profile of FOXC1 in human ocular tissues was determined using quantitative PCR of RNA isolated using a simple and effective procedure for ocular tissue preservation and pigment-free RNA isolation. Higher quality RNA was obtained from human donor eyes preserved in RNAlaterTM compared to RNA extracted from eyes stored on ice or frozen at -80 degrees C. RNA extraction techniques that removed interfering pigment from ocular tissues produced RNA that could be easily amplified by PCR. In the adult human eye, expression of FOXC1 was greatest in the trabecular meshwork (TM) followed by the optic nerve head, choroid/RPE, ciliary body, cornea, and iris. FOXC1 expression levels were much lower in other non-ocular human tissues, such as liver, muscle, lung, heart, and kidney. CONCLUSIONS: Using an optimized donor eye preservation method and tissue RNA isolation procedure, we show that the FOXC1 transcription factor gene, which is known to be associated with developmental glaucoma, also may have an important role in the adult eye.

Aged↗
PubMed2004

A rapid, reliable method to isolate high quality endothelial RNA from small spatially-defined locations.

Phenotypic heterogeneity of the endothelium has important implications for cell sourcing for cardiovascular tissue engineered devices and is fundamental to many cardiovascular diseases. A critical first step to identifying genetic regulators associated with particular endothelial phenotypes is reliable isolation of pure RNA from the cell subpopulations of interest. We present here a rapid method for the isolation of endothelial RNA from small spatially-defined locations, illustrated for two sides of the porcine aortic valve. Endothelial cells were retrieved from fresh tissue by freezing them to a glass substrate, from which they were lysed in guanidine thiocyanate buffer for RNA isolation. Valve endothelial cells isolated by this technique stained positively for CD31 and von Willebrand factor, consistent with an endothelial phenotype, with no evidence of contamination by alpha-smooth muscle actin-positive valve interstitial cells or CD45-positive leukocytes. RNA integrity was excellent in 80% of the samples, with over 100 ng of total RNA typically obtained from each side of the valve. This rapid method yields high quality pure endothelial RNA in sufficient quantities for amplification and subsequent use in device-, cell-, and location-specific transcriptional profiling by microarray technologies, and thus facilitates studies of spatial gene regulation.

Animals↗
PubMed1998

RNA isolation from human skin tissues for colorimetric differential display.

High-quality RNA is essential when analyzing expression patterns of tissues by the differential display technique. However, the isolation of intact RNA can be very difficult when tissues are used that contain many RNAses or that are hard to homogenize (e.g. skin samples). We describe an improved protocol for the extraction of high-quality RNA of snap-frozen biopsies from skin tissues that works in a reproducible and reliable manner. In addition, we developed a simplified non-radioactive differential display technique using RNA from small amounts of skin samples.

Colorimetry↗
PubMed2005

A method for isolating functional RNA from callus of Dendrobium candidum contented rich polysaccharides.

Isolation of high-quality RNA from Dendrobium candidum is particularly difficult. D. candidum contains considerable amounts of polysaccharides that coprecipitate with RNA, which render RNA unsuitable for either cDNA synthesis and/or PCR amplification. In this paper, a rapid and efficient method was described for functional RNA isolation from the callus of D. candidum. The procedure included: (i) an extraction with phenol and isopropyl alcohol, to remove proteins and polyphenols; (ii) purifications by lithium chloride, pre-cooled (-20 degrees C) ethanol successively to remove polysaccharides. The method resulted in high-quality RNA suitable for DDRT-PCR and cDNA library analysis finally.

DNA, Complementary↗
PubMed2005

Feasibility of RNA collection for micro-array gene expression analysis in the treatment of cervical carcinoma: a scientific correlate of RTOG C-0128.

PURPOSE: To determine the feasibility of RNA collection in a multi-institutional cooperative group setting to be utilized for micro-array gene expression analysis, and to describe the methodology. METHODS: RTOG C0128, a phase I-II, protocol was designed to look at the safety and efficacy of external beam radiation therapy to 45 Gy with concomitant 5-FU and cisplatin chemotherapy, brachytherapy to deliver 85 Gy to point A, and Celecoxib at 400 mg twice daily for 1 year. Patients had the option of participating in a tissue collection portion of the protocol to be utilized for micro-array gene expression analysis before treatment and at the time of the first implant. RNA quality was determined by two parameters: the absorbance ratio at 260 nm/280 nm, and by the ratio of the integrated peak of 28S RNA to 18S RNA after gel electrophoresis. RESULTS: From August 2001 to March 2004, 84 patients were accrued to the trial, and tissue was obtained prior to initiation of therapy on 34 patients (40%). FIGO stages for the patients who provided tissue were IB (23%), II (57%), and IIIA-IVA (20%). Additionally, biopsies were obtained at the time of the first implant from 22 of the accrued patients making paired samples available on 26% for RNA extraction and micro-array gene expression analysis. The mean +/- SEM amount of tissue obtained pretreatment was 97 +/- 13 mg compared with 51 +/- 8 mg for tissue obtained at the time of the first implant (P = 0.009). The mean total RNA extracted from the samples prior to treatment was 119 +/- 19 microg versus 35 +/- 6 microg at the time of the first procedure (P = 0.001). The RNA quality was assessed via the absorbance ratio at 260 nm divided by 280 nm. The mean values pretreatment and at first implant were 1.87 +/- 0.07 versus 1.66 +/- 0.11, respectively (P = 0.002); however, the integrated peak of 28S RNA to 18S RNA after gel electrophoresis was not significantly different (P = 0.26). CONCLUSIONS: RNA extraction for gene expression analysis can be successfully performed in the multi-institutional cooperative group setting. Fresh tissue samples were obtained on 40% of accrued patients prior to treatment. The amount of biopsy material and the quantity of RNA extracted were greater prior to treatment compared with the first implant. The quality of RNA was superior prior to treatment as measured by the ratio of absorbance at 260/280 nm. These results indicate that gene expression analysis is feasible in the cooperative group setting utilizing amplification techniques for the RNA. Hopefully, this will allow for improvement in prognosis, therapeutic development, and correlation with acute and late toxicities in patients with cancer.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2005

Optimization of the PAXgene blood RNA extraction system for gene expression analysis of clinical samples.

One major problem associated with collecting whole blood from patients for use as a source of RNA in gene expression studies is that the RNA degrades during collection and storage. Preservation of RNA quality is vital in such studies because the stability of the RNA ultimately affects analysis of gene expression. In this study the PAXgene blood collection system was compared with a standard erythrocyte lysis method for isolating RNA from blood samples. The methods were compared in terms of RNA yield, RNA stabilization, and DNA contamination. The study also included the downstream application to RT-PCR analysis for relative mRNA expression levels of the ribonucleotide reductase subunits R1 and R2. The results show that blood collection in conventional collection tubes, and leukocyte isolation by erythrocyte lysis lead to significant degradation of RNA. Our findings confirm the ability of PAXgene to stabilize RNA in whole blood; however, RNA extracted by the PAXgene method contained significant DNA contamination. Given the low basal expression of the target genes analyzed in this study, contaminating DNA could potentially affect accurate interpretation of RT-PCR data. As a result, the PAXgene protocol was optimized to include off-column DNase treatments, which yielded high-quality RNA suitable for gene expression studies. Furthermore, the results suggest that RNA isolation with PAXgene is advantageous compared to traditional extraction methods for RT-PCR analysis of large or different-sized amplicons.

Actins↗
PubMed2003

Liver gene expression profiles of rats treated with clofibric acid: comparison of whole liver and laser capture microdissected liver.

Clofibric acid (CLO) is a peroxisome proliferator (PP) that acts through the peroxisome proliferator activated receptor alpha, leading to hepatocarcinogenesis in rodents. CLO-induced hepatocarcinogenesis is a multi-step process, first transforming normal liver cells into foci. The combination of laser capture microdissection (LCM) and genomics has the potential to provide expression profiles from such small cell clusters, giving an opportunity to understand the process of cancer development in response to PPs. To our knowledge, this is the first evaluation of the impact of the successive steps of LCM procedure on gene expression profiling by comparing profiles from LCM samples to those obtained with non-microdissected liver samples collected after a 1 month CLO treatment in the rat. We showed that hematoxylin and eosin (H&E) staining and laser microdissection itself do not impact on RNA quality. However, the overall process of the LCM procedure affects the RNA quality, resulting in a bias in the gene profiles. Nonetheless, this bias did not prevent accurate determination of a CLO-specific molecular signature. Thus, gene-profiling analysis of microdissected foci, identified by H&E staining may provide insight into the mechanisms underlying non-genotoxic hepatocarcinogenesis in the rat by allowing identification of specific genes that are regulated by CLO in early pre-neoplastic foci.

Animals↗
PubMed2004

A novel FISH assay for SS18-SSX fusion type in synovial sarcoma.

Synovial sarcoma is a morphologically, clinically and genetically distinct entity that accounts for 5-10% of all soft tissue sarcomas. The t(X;18)(p11.2;q11.2) is the cytogenetic hallmark of synovial sarcoma and is present in more than 90% of the cases. It produces three types of fusion gene formed in part by SS18 from chromosome 18 and by SSX1, SSX2 or, rarely, SSX4 from the X chromosome. The SS18-SSX fusions do not seem to occur in other tumor types, and it has been shown that in synovial sarcoma a clear correlation exists between the type of fusion gene and histologic subtype and, more importantly, clinical outcome. Previous analyses regarding the type of fusion genes have been based on PCR amplification of the fusion transcript, requiring access to good-quality RNA. In order to obtain an alternative tool to diagnose and follow this malignancy, we developed a fluorescence in situ hybridization (FISH) assay that could distinguish between the two most common fusion genes, that is, SS18-SSX1 and SS18-SSX2. The specificity of the selected bacterial artificial chromosome clones used in the detection of these fusion genes, as well as the sensitivity of the analysis in metaphase and interphase cells, was examined in a series of 28 synovial sarcoma samples with known fusion gene status. In all samples, the type of fusion was correctly identified by FISH. Thus, the assay described here should be useful for clarifying unresolved chromosome markers and for identifying fusion gene status in samples from which RNA of sufficient quality for PCR could not be extracted.

Antigens, Neoplasm↗
PubMed2005

Light-induced oxidative stress in choroidal endothelial cells in mice.

PURPOSE: Although light-induced oxidative stress in the retina has been extensively reported, little information regarding light-induced oxidative stress in choroidal endothelial cells (CECs) is available. In the current study, light-induced DNA oxidation and the activation of nuclear factor-kappaB (NF-kappaB), a major oxidative responsive transcription factor, were investigated in mouse CECs. METHODS: Mice were exposed to green light. Light-induced DNA oxidation in CECs was detected by in situ 8-hydroxy-2-deoxyguanosine (8-oxo-dG) immunolabeling. CECs were isolated from the retinal pigment epithelium (RPE)/choroid by using immunomagnetic beads. The isolated CECs were immunochemically characterized by the expression of endothelial markers, CD31, and P1H12. The quality of total RNA from CECs was assessed by a bioanalyzer and RT-PCR. NF-kappaB activation in situ and in isolated CECs was investigated. RESULTS: After a 3-hour exposure to light, the immunoreactivity to anti-8-oxo-dG antibody or anti-NF-kappaB p65 antibody in CECs in situ was significantly increased when compared with unexposed mice. Isolated CECs expressed CD31 and P1H12. The 28S/18S rRNA ratio of RNA isolated from CECs was 1.5:1. CD31 and von Willebrand Factor (vWF) transcripts were predominantly expressed in the RNA from isolated CECs. IkappaBalpha was more heavily phosphorylated in light-exposed than untreated CECs. IkappaBalpha expression levels were increased fivefold in isolated CECs after exposure to light compared to unexposed control subjects. CONCLUSIONS: Exposure to light induces oxidative stress in CECs in vivo. A method for CEC isolation from the mouse RPE/choroid with preservation of RNA quality has been developed. The results of this study may facilitate the ability to identify CEC-specific genes and gene products that respond to photo-oxidative stress.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2006

Optimization of recovery of RNA from formalin-fixed, paraffin-embedded tissue.

Formalin-fixed, paraffin-embedded (FFPE) tissue is the most common specimen available for application of diagnostic assays on tissue after microscopic examination. Not only is there a substantial archive of tissue available, but FFPE tissue remains the best method of preparation for microscopic examination in a routine clinical environment. Molecular assays, especially reverse transcription and polymerase chain reaction and expression array-based assays, offer significant potential as diagnostic, prognostic, and predictive tools, but require high quality RNA. Herein, we have optimized a reliable RNA extraction method for FFPE tissue. It is based on deparaffinization at high temperature coupled with a 3-day lysis at 65 degrees C. The average total RNA yield is 4.5 to 5.5 pg per 1 microm of archival FFPE tissue, and 260/280 ratios are between 1.80 and 1.95. The extracted RNA has a modal fragment length between 100 and 200 nt by the Bioanalyzer analysis. Although modal lengths of RNA fragments were shorter, reverse transcription and polymerase chain reaction was able to amplify amplicons in range of 300 bp. Pretreatment with RNA, later followed by formalin fixation, did not result in improving the RNA quality, but did improve RNA yield. Our method improves the utility of FFPE tissue for molecular profiling studies.

Animals↗
PubMed2000

ROP-1, an RNA quality-control pathway component, affects Caenorhabditis elegans dauer formation.

Caenorhabditis elegans dauer formation is an alternative larval developmental pathway that the worm can take when environmental conditions become detrimental. Animals can survive several months in this stress-resistant stage and can resume normal development when growth conditions improve. Although the worms integrate a variety of sensory information to commit to dauer formation, it is currently unknown whether they also monitor internal cellular damage. The Ro ribonucleoprotein complex, which was initially described as a human autoantigen, is composed of one major 60-kDa protein, Ro60, that binds to one of four small RNA molecules, designated Y RNAs. Ro60 has been shown to bind mutant 5S rRNA molecules in Xenopus oocytes, suggesting a role for Ro60 in 5S rRNA biogenesis. Analysis of ribosomes from a C. elegans rop-1(-) strain, which is null for the expression of Ro60, demonstrated that they contain a high percentage of mutant 5S rRNA molecules, thereby strengthening the notion of a link between the rop-1 gene product and 5S rRNA quality control. The Ro particle was recently shown to be involved in the resistance of Deinococcus radiodurans to UV irradiation, suggesting a role for the Ro complex in stress resistance. We have studied the role of rop-1 in dauer formation. We present genetic and biochemical evidence that rop-1 interacts with dauer-formation genes and is involved in the regulation of the worms' entry into the dauer stage. Furthermore, we find that the rop-1 gene product undergoes a proteolytic processing step that is regulated by the dauer formation pathway via an aspartic proteinase. These results suggest that the Ro particle may function in an RNA quality-control checkpoint for dauer formation.

Animals↗
PubMed1995

Identification of an EWS-pseudogene using translocation detection by RT-PCR in Ewing's sarcoma.

The presence of a t(11;22)(q24;q12) translocation is one of the characteristic features of the Ewing family of tumors. The detection of the fusion gene product by RT-PCR using primers at both sides of the breakpoints has been advocated as a diagnostic tool. By applying this technique appropriate internal controls are required. We found that the use of normal non-rearranged EWS mRNA as an internal control for RNA quality may lead to conflicting data. We obtained PCR products of the expected size for the normal EWS mRNA in both RNA and DNA samples, suggesting, the existence of one or more EWS pseudogenes. A 109 bp sequence at the 5' end of this PCR-product contained a correctly spliced exon junction and was 97% homologous to the EWS cDNA sequence. Similarly two such junctions were found in a 346 bp sequence of the 3' end, which was 89% homologous. Hence EWS should not be used as an internal control for the RNA quality in a RT-PCR based test for the presence of the translocation.

Base Sequence↗
PubMed2006

An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development.

BACKGROUND: Accuracy in quantitative real-time RT-PCR is dependent on high quality RNA, consistent cDNA synthesis, and validated stable reference genes for data normalization. Reference genes used for normalization impact the results generated from expression studies and, hence, should be evaluated prior to use across samples and treatments. Few statistically validated reference genes have been reported in grapevine. Moreover, success in isolating high quality RNA from grapevine tissues is typically limiting due to low pH, and high polyphenolic and polysaccharide contents. RESULTS: We describe optimization of an RNA isolation procedure that compensates for the low pH found in grape berries and improves the ability of the RNA to precipitate. This procedure was tested on pericarp and seed developmental series, as well as steady-state leaf, root, and flower tissues. Additionally, the expression stability of actin, AP47 (clathrin-associated protein), cyclophilin, EF1-alpha (elongation factor 1-alpha), GAPDH (glyceraldehyde 3-phosphate dehydrogenase), MDH (malate dehydrogenase), PP2A (protein phosphatase), SAND, TIP41, alpha-tubulin, beta-tubulin, UBC (ubiquitin conjugating enzyme), UBQ-L40 (ubiquitin L40) and UBQ10 (polyubiquitin) were evaluated on Vitis vinifera cv. Cabernet Sauvignon pericarp using three different statistical approaches. Although several of the genes proved to be relatively stable, no single gene outperformed all other genes in each of the three evaluation methods tested. Furthermore, the effect of using one reference gene versus normalizing to the geometric mean of several genes is presented for the expression of an aquaporin and a sucrose transporter over a developmental series. CONCLUSION: In order to quantify relative transcript abundances accurately using real-time RT-PCR, we recommend that combinations of several genes be used for normalization in grape berry development studies. Our data support GAPDH, actin, EF1-alpha and SAND as the most relevant reference genes for this purpose.

Aquaporins↗
PubMed2006

Biobanking of fresh frozen tissue: RNA is stable in nonfixed surgical specimens.

Molecular tools for tissue profiling, such as expression microarrays and real-time PCR, generally require collection of fresh frozen tissues as sources of high-quality RNA. The fragile nature of RNA prompted us to examine the effects of storage time and transport conditions with regard to RNA integrity and gene expression in nonfixed surgical human specimens. At surgery, fresh normal tonsil and colon tissue was cut into pieces and snap frozen. Additional fresh tissue pieces were (i) left at room temperature, (ii) kept on ice, (iii) in normal saline or (iv) in a commercial RNA-stabilizing buffer (RNAlater) and snap frozen after 0.5, 1, 3, 6 and 16 h. Structural RNA integrity was analysed by microchip electrophoresis. Surprisingly, RNA remained stable in both tissue types under all conditions tested for up to 6-16 h. Gene expression by real-time PCR of cfos, HIF1alpha, Bcl2, PCNA, TGFbeta1 and SMAD7 was analysed at different storage time points in tonsil tissue. Expression levels were essentially stable when samples were kept on ice, while marked regulation of single genes was observed during storage at room temperature, in normal saline and in RNAlater. Furthermore, we analysed selected tissue types from the local biobank representing 47 normal and malignant tissues transported on ice for up to 2-3 h before biobanking. RNA prepared from 45 of the 47 samples exhibited distinct ribosomal peaks indicating intact RNA. This study shows that RNA degradation is a minor problem during handling of fresh human tissue before biobanking. Our data indicate that nonfixed tissue specimens may be transported on ice for hours without any major influence on RNA quality and expression of the selected genes. However, further studies are warranted to clarify the impact of transport logistics on global gene expression.

Base Sequence↗
PubMed2006

Extraction of high-quality, melanin-free RNA From Mycosphaerella fijiensis for cDNA preparation.

High-quality RNA preparations are critical for further applications such as reverse transcriptase-polymerase chain reaction (RT-PCR) transcript amplifications, and elaboration of cDNA and expressed sequence tag libraries. Melanins are phenolic compounds present in many fungi and apparently play key roles in fungi pathogenesis and survival. However, during RNA extraction these compounds constitute a significant challenge to extraction of substantial quantities of high-quality RNA, and consequently to preparation of cDNA libraries. No method currently exists for RNA extraction from Mycosphaerella fijiensis that produces high quantities of melanin-free RNA. This fungus is the most important pathogen of cultivated Musa sp. varieties. A comparison is made between results obtained from the Trizol and RNeasy protocols for RNA extraction, two commercially available methods commonly used to obtain RNA from various sources. An improved methodology is described that allows isolation of intact RNA and elimination of melanins from M. fijiensis mycelium. RNA quality is evaluated by electrophoresis in formaldehyde-agarose gels, RT into cDNAs, and subsequent PCR amplification using primers designed against actin and beta- tubulin from fungi.

Ascomycota↗