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At least 199 records · Page 11Linked to original sources
PubMed2006

Validation of two reference genes for mRNA level studies of murine disease models in neurobiology.

Reverse transcription of extracted cellular RNA combined with real-time PCR is now an established method for sensitive detection and quantification of specific mRNA level changes in experimental models of neurological diseases. To neutralize the impact of experimental error and make quantification more precise, normalization of test gene data using data from a constantly expressed gene, a reference gene that is tested along with the test gene, is required. There is no single gene constantly expressed under all experimental conditions. For a given set of conditions or a given disease model, identification of an unaffected reference gene is necessary. In this report, we present our findings from evaluation and validation of the genes encoding hypoxanthine guanine phosphoribosyl transferase 1 (HPRT1) and glyceraldehyde phosphate dehydrogenase (GAPDH) as individual reference genes in mRNA level studies involving four murine neurological disease models. We find both genes are suitable as a reference gene with these four models, provided quantification of subtle changes are avoided. We furthermore demonstrate that above a certain threshold of test mRNA level changes and given high quality RNA processing, normalization to total RNA alone provides for equally reliable quantitative mRNA level results.

Animals↗
PubMed2001

Single-tube gene-specific expression analysis by high primer density multiplex reverse transcription.

Molecular genetics is rapidly moving from simple identification of a gene of interest to characterization of gene products as components in complex networks. Critical tools for gene product analysis require a rapid method for evaluation of contextual expression. Here, we describe a robust, high primer density, single-tube, multiplex reverse transcription (HD-MRT) technique. This approach is capable of analyzing for the presence of numerous transcripts when polymerase chain reaction (PCR) is subsequently employed for individual gene-specific sequence amplification (HD-MRT-PCR). This assay substantially increases the total number of different cDNAs for amplification beyond previously published techniques. Our approach simultaneously eliminates RNA quality control issues for samples run in parallel while improving efficiency in the use of time and materials. This assay is designed for broad applicability and accessibility, employs modifications of commercially available components, and allows more than 25 independently selected gene-specific primers to be used simultaneously. Our protocol allows multiplexed primers to behave similarly to uniplex RT reactions, while avoiding potential interference between gene-specific and/or nonspecific primers during annealing and reverse transcription. Expression of putatively networked homologous transcripts was analyzed in multiple cell lines and tissues from mouse and human to validate the technique.

Animals↗
PubMed2006

Ultrasonic decalcification offers new perspectives for rapid FISH, DNA, and RT-PCR analysis in bone marrow trephines.

The requisite analyses on bone marrow biopsies are increasing: Molecular analyses such as fluorescence in situ hybridization (FISH), polymerase chain reaction (PCR), and reverse transcriptase (RT)-PCR are demanded in addition to morphology and immunohistochemistry to improve diagnostic accuracy. Moreover, analysis of certain molecular prognostic or predictive biomarkers is increasingly mandatory in the assessment of hematologic diseases. In some circumstances, only formalin fixed, bone-containing tissue is available for molecular analysis. Because various fixation and decalcification procedures can impair DNA and RNA quality, there is an urgent need for standardized decalcification protocols which allow FISH and PCR analysis. In this study we developed a routinely applicable decalcification protocol to optimize the molecular analysis method although preserving morphology and immunohistochemical results. Therefore, we compared 2 different approaches including ultrasonic decalcification versus nonultrasonic procedures and ethylenediaminetetraacetate-based reagents versus acid-based ones. In our hands, the combined use of ultrasound and ethylenediaminetetraacetate-based reagents permits successful interphase FISH, PCR, and RT-PCR analysis whereas concomitantly preserving morphology and antigeneicity.

Biopsy↗
PubMed2001

Pre-clinical validation of a novel, highly sensitive assay to detect PML-RARalpha mRNA using real-time reverse-transcription polymerase chain reaction.

We have developed a sensitive and quantitative reverse-transcription polymerase chain reaction (RT-PCR) assay for detection of PML-RARalpha, the fusion oncogene present as a specific marker in >99% of cases of acute promyelocytic leukemia (APL). The assay is linear over at least 5 orders of magnitude of input DNA or RNA, and detects as few as 4 copies of PML-RARalpha plasmid DNA. PML-RARalpha transcripts could be detected in mixtures containing 2 to 5 pg of RNA from fusion-containing cells in a background of 1 microg of RNA from PML-RARalpha-negative cells. Using 1.0 to 2.5 microg of input RNA, the sensitivity of the assay was between 10(-5) and 10(-6). Furthermore, determination of GAPDH copy number in each reaction allowed an accurate assessment of sample-to-sample variation in RNA quality and reaction efficiency, with consequent definition of a detection limit for each sample assayed. Using an internal calibrator, assay precision was high, with coefficients of variation between 10 and 20%. An interlaboratory study using coded samples demonstrated excellent reproducibility and high concordance between laboratories. This assay will be used to test the hypothesis that sensitive and quantitative measurement of leukemic burden, during or after therapy of APL, can stratify patients into discrete risk groups, and thereby serve as a basis for risk-adapted therapy in APL.

Biomarkers, Tumor↗
PubMed2007

The molecular basis of urgency: regional difference of vanilloid receptor expression in the human urinary bladder.

AIM: Treatments targeting vanilloid receptor TRPV1 are effective in some bladder disorders. Our aim was to determine the expression profiles of TRPV1 in regions of human bladder and test the hypothesis that there would be an upregulation of TRPV1 in mucosa of patients with bladder hypersensitivity but not idiopathic detrusor overactivity (IDO). MATERIALS AND METHODS: Women with sensory urgency (SU), interstitial cystitis (IC), and IDO were investigated by videourodynamics and cystoscopy. Control biopsies were used for comparison. Biopsies were dissected into mucosa and muscle, and evaluated for TRPV1 mRNA expression using quantitative competitive RT-PCR (QC-RT-PCR). RESULTS: TRPV1 mRNA from SU trigonal mucosa was significantly higher than control trigonal mucosa or SU bladder body mucosa. In contrast, in IDO patients, there was no difference between trigonal mucosa and body mucosa. In IC biopsies, RNA quality was substandard and unable to be used for analysis. The most striking finding was that TRPV1 mRNA expressed in SU trigonal mucosa was significantly inversely correlated with the bladder volume at first sensation of filling during cystometry. No such relationship was seen for IDO trigonal mucosa. No difference was seen in bladder body mucosa from any disease groups compared with age-matched control. CONCLUSIONS: The symptoms of SU were associated with the increased expression of TRPV1 mRNA in the trigonal mucosa. No upregulation or regional differences of TRPV1 mRNA were seen in IDO patients. TRPV1 may play a role in SU and premature first bladder sensation on filling.

Adult↗
PubMed2004

Differential modulation of gene expression among rat tissues with warm ischemia.

The aim of this study is to determine if warm ischemia after surgical extirpation impacts gene expression in tissue samples which will be used for cDNA array analysis. We investigated effects of warm ischemia on gene expression in lung, liver, kidney, and spleen of rats, chronologically, using an original cDNA array, real-time quantitative RT-PCR and immunohistochemistry. Although no visible alteration was found in RNA quality, cDNA array showed that expression of many genes was modulated by warm ischemia within 60 min in these tissues, 19.1% of the tested genes in lung, 11.0% in liver, 5.1% in kidney, and 16.2% in spleen. Quantitative RT-PCR revealed that warm ischemia significantly induced up-regulation of immediate early genes, c-fos, Egr-1, and c-jun, in lung, but not in liver. These findings suggest that genes may show tissue-dependent differential transcriptional response against warm ischemia. Tissue samples obtained from patients during surgery cannot completely escape effects of ischemia. In case of examination by cDNA array analysis, biologists should keep in mind that tissue samples come equipped with particular footprints.

Animals↗
PubMed1999

Rapid and sensitive minimal residual disease detection in acute leukemia by quantitative real-time RT-PCR exemplified by t(12;21) TEL-AML1 fusion transcript.

Because previous PCR-based methodologies for detection of minimal residual disease (MRD) in leukemia patients have been too cumbersome to allow for widespread clinical usefulness, we have employed a real-time quantitative PCR (RQ-PCR) system to develop an MRD assay for t(12;21). We initially determined the expression of the different alternatively spliced TEL-AML1 mRNAs found in t(12;21) breakpoint variants I and II. We then optimized PCR primers for the RQ-PCR system and, using the t(12;21)+ REH cell line in spiking experiments, found a linear detection of TEL-AML1 over at least five logs. Moreover, 1 malignant cell in a background of 1,000,000 normal cells could be detected. The expression of the GAPDH, ABL, and beta(2)-microglobulin (beta2M) housekeeping genes were then compared in normal donors and in leukemic patients, and the very stably expressed beta2M was selected as an internal reference gene, allowing us to compensate for variation in RNA quality and day-to-day variation. In 12 samples from t(12;21)-positive patients at diagnosis, the levels of the TEL-AML1 fusion transcripts were found to vary up to 14-fold after normalization to beta2M. Interestingly, in samples obtained from seven patients at diagnosis, during induction chemotherapy, or relapse, the level of TEL-AML1 in peripheral blood (PB) and bone marrow (BM) was found to differ only by threefold, suggesting that MRD may be evaluated in PB samples in most patients. We conclude that this assay could set new standards for t(12;21) MRD detection with its accuracy, its high throughput, and its short turnover time for samples. Genes Chromosomes Cancer 26:355-365, 1999.

Bone Marrow↗
PubMed1997

Field cancerisation and polyclonal p53 mutation in the upper aero-digestive tract.

Field cancerisation of the aerodigestive tract is caused by chronic exposure to alcohol and tobacco, but the nature of the genetic alterations preceding overt malignancy is unknown. To identify potential field changes we have used a functional assay which tests the transcriptional competence of human p53 expressed in yeast. To increase the sensitivity and reliability of the technique for samples containing under 20% mutant p53, the 5' and 3'-ends of the p53 cDNA were examined separately. With this split form of the assay the tissue p53 mRNA acts as its own control for RNA quality. Mutations were detected in 87% (46/53) of tumours, reflecting the high sensitivity of the technique. Multiple biopsies of histologically normal tissue from the upper aero-digestive tract were tested and clonal p53 mutations were identified in 76% (38/50) of biopsies from patients presenting with multiple tumours compared with 32% (38/117) of biopsies from patients presenting with single tumours (P<0.000001). All patients (16/16) presenting with multiple tumours had at least one positive biopsy, compared with only 53% (19/36) of patients presenting with single tumours (P <0.001). This defines expansion of multiple clones of mutant p53-containing cells as an important biological mechanism of field cancerisation, and provides a means to identify patients likely to benefit from intensive screening for the development of new head and neck tumours.

Adult↗
PubMed2002

Adrenocorticotrophic hormone (ACTH) stimulation of sheep fetal adrenal cortex can occur without increased expression of ACTH receptor (ACTH-R) mRNA.

In the present study, it was hypothesized that the adrenocorticotrophin hormone receptor (ACTH-R) would be up-regulated in the adrenal gland of the sheep fetus following infusion of physiological amounts of ACTH, as shown for adrenal cortical cells in culture. In chronically catheterized sheep, an intravenous infusion of ACTH(1-24) was given to 6 fetuses for 24 h at a rate of 0.5 microg h(-1), starting on Day 126 or 127 of gestation (term approximately 147 days). Four control fetuses received an infusion of vehicle (saline). Total RNA was extracted from the fetal adrenal glands by the guanidinium thiocyanate method. Expression of specific mRNAs was determined by ribonuclease protection assay using cRNA probes directed against: ACTH-R; the steroid enzymes side-chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), 17apha-hydroxylase (P450c17) and 21beta-hydroxylase (P450c21); and beta-actin. Ratios of mRNA expression to beta-actin mRNA expression (arbitrary units) were calculated to correct for differences in RNA quality between samples. The concentration (mean +/- SEM) of immunoreactive cortisol in fetal plasma was greater after ACTH infusion than after vehicle infusion (47 +/- 3 v. 13 +/- 2 ng mL(-1) respectively; P<0.001). Adrenal expression of P450scc and P450c21 mRNA increased after ACTH infusion (P<0.05), whereas expression of P450c17 and 3beta-HSD mRNA was unchanged. There was no difference in ACTH-R mRNA expression between ACTH- and vehicle-infused fetuses (254 +/- 48 v. 305 +/- 76 arbitrary units respectively). It was concluded that ACTH is able to increase plasma cortisol concentrations in the sheep fetus by up-regulating cortisol synthesis in the adrenal gland, but that in vivo this does not require up-regulation of ACTH-R mRNA.

3-Hydroxysteroid Dehydrogenases↗
PubMed2026

Stage-Independent Real-Time Subtype Classification and Comprehensive Biopsy Profiling of Urothelial Carcinomas by the Lund Taxonomy System.

Bladder cancer is a heterogeneous malignancy with diverse clinical outcomes, and conventional pathological assessment alone is insufficient to capture its underlying biology. Gene expression profiling can stratify tumors into molecular subtypes with prognostic and predictive potential, but the reliability of transcriptomic classification and its clinical utility remains to be established. The translational/observational UROSCANSEQ study (ISRCTN15459149) prospectively evaluates RNA-based Lund Taxonomy (LundTax) molecular subtype classification in a clinical setting. Among 784 consecutive biopsies collected between 2018 and 2022, RNA sequencing was successful for 90% of all biopsies, encompassing 662 bladder cancer patients with a stage distribution of 48% Ta, 27% T1, 24% &#x2265;T2, and 1% CIS. We demonstrate that the LundTax subtype classification algorithm, applied to individual samples, accurately identifies cancer cell phenotypes with characteristic gene and protein expression patterns in a manner robust to RNA quality, data preprocessing strategies, and batch effects, supporting its clinical feasibility across both non-muscle-invasive and muscle-invasive disease. We further extend the LundTax framework by incorporating single-sample molecular risk scores reflecting tumor grade, proliferation, and progression risk, as well as tumor microenvironment signatures. Both risk scores and overall immune and stromal content in biopsies were significantly associated with an increased risk of clinical progression in noninvasive disease. In a separate analysis of the relative cellular composition of the tumor microenvironment, however, only the fraction of natural killer cells remained significant. Together, the expanded LundTax system provides a comprehensive molecular portrait of individual tumor biopsies. By explicitly separating cancer cell-intrinsic phenotypes, prognostic indexes, and microenvironmental signals, the framework minimizes biological confounding and establishes a strong foundation for future studies evaluating clinical outcomes and treatment responses.

Humans↗
PubMed2006

The majority of Escherichia coli mRNAs undergo post-transcriptional modification in exponentially growing cells.

Polyadenylation of RNAs by poly(A) polymerase I (PAP I) in Escherichia coli plays a significant role in mRNA decay and general RNA quality control. However, many important features of this system, including the prevalence of polyadenylated mRNAs in the bacterium, are still poorly understood. By comparing the transcriptomes of wild-type and pcnB deletion strains using macroarray analysis, we demonstrate that >90% of E.coli open reading frames (ORFs) transcribed during exponential growth undergo some degree of polyadenylation by PAP I, either as full-length transcripts or decay intermediates. Detailed analysis of over 240 transcripts suggests that Rho-independent transcription terminators serve as polyadenylation signals. Conversely, mRNAs terminated in a Rho-dependent fashion are probably not substrates for PAP I, but can be modified by the addition of long polynucleotide tails through the biosynthetic activity of polynucleotide phosphorylase (PNPase). Furthermore, real-time PCR analysis indicates that the extent of polyadenylation of individual full-length transcripts such as lpp and ompA varies significantly in wild-type cells. The data presented here demonstrates that polyadenylation in E.coli occurs much more frequently than previously envisioned.

Escherichia coli↗
PubMed2006

Molecular monitoring of chronic myelogenous leukemia: identification of the most suitable internal control gene for real-time quantification of BCR-ABL transcripts.

Monitoring breakpoint cluster region-Abelson kinase (BCR-ABL) levels in patients treated for chronic myelogenous leukemia (CML) has become an integral part of patient management. Real-time reverse transcriptase-polymerase chain reaction is the method of choice for this purpose because of its high analytical sensitivity and reproducibility. Given the variation of RNA quality and quantity in clinical specimens, accurate quantitative assessment of BCR-ABL depends on normalization of the BCR-ABL signal to an appropriate internal reference. However, the controls used by different laboratories vary, and there is no clear consensus on an ideal reference due to limited investigations. In this study, we compared nine commonly used control genes for three criteria: mRNA abundance, levels in CML and non-CML cells, and their degradation kinetics in comparison with BCR-ABL. We found that beta-glucuronidase (GUSB) is the most suitable among the nine genes tested. Although ABL is most widely used, our data suggest that the amount of ABL is different in CML and non-CML cells. Moreover, ABL levels are regulated by cellular stress. These findings have a direct impact on current clinical laboratory practice and patient care because the use of a proper control gene affects the reported levels of BCR-ABL transcripts used for patient management decisions.

Adult↗
PubMed1998

The mRNA expression of cytokines and their receptors in cultured iris pigment epithelial cells: a comparison with retinal pigment epithelial cells.

It has been suggested that human iris pigment epithelial (IPE) cells isolated from iridectomized tissue could be used as autologous cells for transplantation into the subretinal space in diseases with dysfunctional retinal pigment epithelium (RPE). RPE cells synthesize a number of cytokines and their receptors which are important for its proper function. Nearly nothing is known about the capacity of IPE to synthesize cytokines or responding to them. To compare the mRNA expression of 36 cytokines or their receptors in cultured adult IPE cells and RPE cells we used semi-quantitative reverse transcription polymerase chain reactions (RT-PCR). Included in our assay were cytokines with known expression in RPE to get a broad basis for comparing IPE cells: basic fibroblast growth factor (bFGF or FGF-2), and one of its receptor (FGFR-1), epidermal growth factor (EGF), and its receptor EGF-R, transforming growth factor beta(TGFbeta), and its type III receptor TGFbeta-R3, the platelet-derived growth factors and receptors (PDGF A, PDGF B, PDGF-Ralpha, PDGF-Rbeta), tumor necrosis factor alpha(TNFalpha), and two receptors TNF-R1 and TNF-R2, insulin (INS) with receptor INS-R, insulin-like growth factors (IGF1, IGF2), and receptors (IGF1-R, IGF2-R), vascular endothelial growth factor (VEGF), and two receptors (VEGF-R1 or FLT-1 and VEGF-R2 or FLK-1), the receptor for VEGF-C: VEGF-R3 or FLK-4, interleukin 6 (IL6), and its receptor (IL6-R), nerve growth factor (NGF), interleukin 1alpha(IL1alpha), and a receptor (IL1-R). In addition, cytokines or their receptors not known to be expressed in RPE were included to widen our picture of cytokine gene expression in the eye: stem cell factor (SCF), its receptor (SCF-R), low-affinity nerve growth factor receptor p75 (p75(NGF-R), ciliary neutrothropic factor (CNTF), and its receptor (CNTF-R), glycoprotein 130 interleukin 6 transducer (gp130 (IL6-SD), leukemia inhibitory factor (LIF), and its receptor (LIF-R). Semi-quantitative expression data were obtained using series of fivefold dilutions of each cDNA and a fixed number of PCR cycles. The expression of RPE 65, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta2-microglobulin (B2MG) was used as a control for cellular origin, RNA quality and PCR conditions. With the exception of insulin and tumor necrosis factor alphaall other cytokines analysed and their receptors were expressed in both IPE and RPE cells, even though the levels varied. No qualitative or quantitative difference were observed in the mRNA expression level of 34 (94%) of the cytokines or receptors between IPE and RPE. In contrast, the mRNA expression level of vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor 2 [VEGF-RS (FLK-1)] was lower in IPE than in RPE cells. As an increased expression of VEGF in the RPE in maculae with age-related macular disease could be involved in its pathogenesis, a decreased expression of angiogenic growth factors in IPE cells could possibly be beneficial for the therapy of age-related maculopathy if indeed other tasks of non-functional RPE cells could be performed by IPE cells. The similarity of the mRNA expression pattern in 94% of the cytokines analyzed supports the assumption that IPE cells potentially can perform functions of RPE cells in the appropriate environment.

Adult↗
PubMed2005

Application of an electric DNA-chip for the expression analysis of bioprocess-relevant marker genes of Bacillus subtilis.

The knowledge of critical process-relevant genes can be used for an improved control of bioprocesses. So far bioprocess-relevant marker genes can be analyzed by established expression analysis methods only off-line. In this study, an alternative approach for a potential at-line monitoring of gene expression during bioprocesses is suggested. This approach is based on the measurement of specific mRNAs on an electric DNA-chip in connection with a magnetic bead-based sandwich hybridization. In order to allow an at-line measurement of specific mRNAs an improved method for a fast and partially automated isolation of high quality-RNA samples was developed. The expression analysis of the electric DNA-chip was compared with optical DNA micro arrays and the real time RT-PCR for three selected process-relevant genes of Bacillus subtilis. We demonstrate that the mRNA analysis by means of the electric DNA-chip gives similar results compared to the micro array analysis and the real time RT-PCR technique.

Bacillus subtilis↗
PubMed1987

Construction of cDNA libraries by blunt-end ligation: high-frequency cloning of long cDNAs from filamentous fungi.

A simplified cDNA synthesis and cloning method, suitable for efficient generation of cDNA libraries at frequencies up to 10(6) clones/micrograms mRNA, is described. Routine synthesis of transcripts of well over 4 kb is facilitated by the use of high-quality RNA template isolated from materials rich in RNases. Laborious cloning steps, like tailing or addition of linkers, can be omitted by the use of efficient blunt-end ligation to plasmid vectors, and rapid verification as well as characterization of the clones is possible by double-stranded plasmid sequencing. Using this method we have constructed several cDNA libraries of different filamentous fungi and show here the synthesis and cloning of cDNA copies larger than 1.8 kb corresponding to three Trichoderma reesei cellulases.

Bacteriophage lambda↗
PubMed2004

Characterization of an animal model of spontaneous congenital unilateral obstructive uropathy by cDNA microarray analysis.

INTRODUCTION: The pathophysiology of congenital obstructive uropathy has been studied intensively in several animal models. A reliable parameter for early detection of relevant obstruction is not yet identified. For further investigation of the complex regulatory events of obstructive uropathy, we used a cDNA microarray technology for a parallel differential expression analysis of about 15000 different genes of obstructed, contralateral and healthy kidneys of rats with spontaneous congenital obstructive uropathy. METHODS: Total cellular RNA of obstructed, contralateral and healthy control kidneys of 32-35 days old rats was extracted and pooled. RNA quality and quantity was assessed using a Bioanalyzer 2100. mRNA expression for selected marker genes was assessed by real time PCR. High throughput gene expression profiling was performed with cDNA microarrays with differentially labeled cDNA targets. RESULTS: Beside expected typical alterations of several growth factors such as TGF-beta, EGF, IGF-1, PDGF, we observed overexpression of extracellular matrix proteins and a decreased activity of antioxidant enzymes. Additionally, NFkappaB, a nuclear transcription factor involved in the development of interstitial fibrosis, was slightly up-regulated in the obstructed kidneys. Down-regulation of genes involved in tubular sodium transport indicated impaired concentration ability of the obstructed kidneys. Furthermore, we found up-regulation of pro-apoptotic genes including transcripts for the proapoptotic protein Siva. Interestingly, TNFalpha, another factor involved in the pathophysiology of congenital obstructive uropathy, was not differentially regulated. CONCLUSIONS: The alterations of the genes for growth factors, extracellular matrix proteins, antioxidant enzymes, sodium transport and genes involved in apoptosis support the representative character of this animal model for congenital obstructive uropathy. In the rather advanced stage of congenital renal obstruction a possible explanation of the lacking differential regulation of TNFalpha highlights it as a possible marker of earlier stages of obstruction. Furthermore, the possible involvement of the Siva/CD27 pathway in the apoptotic cascade also offers a new possibility to find a sensitive marker to detect renal obstruction already in an earlier stage.

Analysis of Variance↗
PubMed2001

Expression profiling of ductal carcinoma in situ by laser capture microdissection and high-density oligonucleotide arrays.

Gene expression profiling through the use of nucleic acid arrays is a powerful method for the molecular classification of human neoplasms. Laser capture microdissection is an equally useful technique to selectively isolate defined cell populations from heterogeneous histological tissue sections. In this report, we demonstrate how a modest use of laser capture microdissection is sufficient to isolate nanogram quantities of high-quality RNA. Together with the use of several internal standards and microcapillary electrophoresis of input RNA, two rounds of linear molecular amplification have been used to generate sufficient quantities of labeled target for hybridization to high-density oligonucleotide expression arrays. Results demonstrate that the technique is reproducible, generates only modest biasing of the original transcript population, and is comparable to the sensitivity achieved with standard methodology. Using this approach, we have compared the expression profiles of nonmalignant human breast epithelium and adjacent ductal carcinoma in situ lesions from breast cancer patients. Several genes, previously implicated in human breast cancer progression, demonstrate differential expression among the microdissected cell populations.

Breast↗
PubMed2006

Tissue preparation and banking.

With the increasing application of genomic and proteomic technologies to the research of neurological and psychiatric disorders it has become imperative that the postmortem tissue utilized be of the highest quality possible. Every step of the research design, from identifying donors, acquiring sufficient information for accurate diagnosis, to assessing tissue quality has to be carefully considered. In order to obtain high-quality RNA and protein from the postmortem brain tissue a standardized system of brain collection, dissection, and storage must be employed and key ante- and postmortem factors must be considered. Reliable RNA expression and protein data can be obtained from postmortem brains with relatively long postmortem intervals (PMIs) if the agonal factors and acidosis are not severe. While pH values are correlated with RNA integrity number (RIN), a higher pH does not guarantee intact RNA. Consequently RNA integrity must be assessed for every case before it is included in a study. An analysis of anti- and postmortem factors in a large brain collection has revealed that several diagnostic groups have significantly lower pH values than other groups, however, they do not have significantly lower RIN values. Moreover, the lower pH of these groups is not entirely due to agonal factors and/or smoking, indicating that these subjects may have additional metabolic abnormalities that contribute to the lower pH values.

Autopsy↗