A time course study demonstrating RNA stability in postmortem skin

Knowledge of the factors regulating the rate of mRNA degradation, including postmortem delay, is important in determining the reliability of gene expression patterns in dermal tissue. Since RNA stability can be tissue dependent, this study evaluates the effect of postmortem interval on the integrity of total RNA or the levels of representative mRNA species in murine cutaneous tissue. Pieces of fresh skin tissue were excised for periods of 0-60 min from SKH-1 female hairless mice that were maintained at room temperature post-sacrifice. Total RNA was subsequently isolated and RNA integrity from each specimen was evaluated. Bioanalyzer profiles showed no apparent change in 28S/18S rRNA ratio or RNA integrity number at time points up to 60 min. Changes in mRNA expression levels of five selected genes were determined by real-time quantitative PCR. There were no statistical differences in the relative gene expressions of Ccnd1, Hif1alpha, cMyc and Cyr61 as a function of postmortem interval. Our data suggest that the molecular quality of cutaneous tissue is well preserved for at least 60 min after death, which can be regarded as important information for consideration of the order for tissue procurement in in vivo studies and acute ex vivo dermal studies.     

The relative importance of premortem acidosis and postmortem interval for human brain gene expression studies: selective mRNA vulnerability and comparison with their encoded proteins

To help account for the variable quality and quantity of RNA in human brain, we have studied the effect of premortem (agonal state) and postmortem factors on the detection of poly(A)⁺mRNA and eight mRNAs. For comparison, the influence of the same factors upon gene products encoded by the mRNAs was studied immunocytochemically or by receptor autoradiography. Brain pH declined with increasing age at death and was related to agonal state severity, but was independent of postmortem interval and the histological presence of hypoxic changes. By linear regression, pH was significantly associated with the abundance of several of the RNAs, but not with poly(A)⁺mRNA, immunoreactivities, or binding site densities. Postmortem interval had a limited influence upon mRNA and protein products. Freezer storage time showed no effect. Parallel rat brain studies showed no relationship between postmortem interval (0–48 h) and amounts of total RNA, poly(A)⁺RNA, or two individual mRNAs; however, RNA content was reduced by 40% at 96 h after death. pH is superior to clinical assessments of agonal state or mode of death in predicting mRNA preservation. It provides a simple means to improve human brain gene expression studies. pH is stable after death and during freezer storage and can be measured either in cerebrospinal fluid or in homogenised tissue.     

人脑组织库样本100例质量及影响因素分析

目的检测人脑组织库中保存的脑组织蛋白质、DNA及RNA质量,分析影响其质量的主要因素。方法选取中国医学科学院北京协和医学院人脑组织库保存的100例全脑组织样本,提取蛋白质、DNA及RNA,测定其浓度及吸光度,电泳检测完整性,用Western blotting及Rael-time PCR分别检测。根据不同年龄、死亡原因、取材延迟时间以及样本保存时间进行分组,统计学分析影响脑组织质量的因素。结果人脑组织库中大部分脑组织的DNA、RNA和蛋白质质量保存完好,不同年龄、死亡原因对其质量无显著影响。取材延迟时间超过24h者,其蛋白质浓度及RNA浓度及质量有显著下降。样本保存时间对蛋白质及DNA质量无显著影响,但保存1年以上可能对RNA浓度有影响。结论进行RNA相关分析的人脑组织标本取材延迟时间最好控制在8 h之内,-80℃环境下保存1年之内为佳。而DNA和蛋白质相关分析的时间可以适当延长。     

Assessing RNA quality in postmortem human brain tissue

The development of microarrays for the screening of gene expression has highlighted the importance of obtaining high quality RNA. We have investigated whether it was possible to obtain RNA of sufficiently good quality from postmortem human tissue using samples obtained from the New Zealand Neurological Foundation Human Brain Bank. We have investigated the effect of PM delay, the duration of the agonal state, the tissue pH, the age at death and the sex of the patient on the quality of normal human brain tissue and tissue from patients with various neurodegenerative conditions. Postmortem delay was shown to affect the RNA quality adversely, but the magnitude of the effect was small. While cerebellar RNA quality was not always an exact predictor of the RNA quality in other brain regions of interest, it was shown to have some predictive value and can be used as a preliminary indicator. The principle finding was that RNA quality is most strongly affected by the pH of the tissue, with both the pH and the RNA quality being influenced by the mode of death.     

Recovery and measurement of specific RNA species from postmortem brain tissue: a general reduction in Alzheimer's disease detected by molecular hybridization

The recovery of RNA from postmortem (PM) brain tissues was quantified by molecular hybridization. RNA degradation rates postmortem were faster in mice with herpes encephalitis than with uninfected mice, but clear ribosomal peaks could be seen up to 72 hr after death. In a comparison between frontal cortex samples from neurologically normal and Alzheimer's disease cases a reduction in ribosomal, poly A, preproenkephalin, and preprosomatostatin RNA levels was observed in the Alzheimer's disease group. This general reduction may be influenced by the cause of death as well as the pathology.     

Impact of fixative on recovery of mRNA from paraffin-embedded tissue.

Due to the evolution of advanced tissue-analysis tools, such as proteomics and functional and structural genomics, the demands for handling and preserving samples are changing. For gene expression analysis, the presence of intact and extractable messenger RNA in the test material is mandatory. To find an optimal fixative for tissues aimed for such analyses, we evaluated the morphology-, protein antigen-, and RNA-maintaining abilities of 2 precipitating tissue fixatives, methanol-acetone and Carnoy's. Both fixatives preserved the morphology and protein epitopes of tissues and allowed extraction of total RNA that was of significantly higher quality than RNA extracted from formalin-fixed tissue. Carnoy's fixative performed better than methanol-acetone in maintaining the integrity of RNA, especially when the fixed, paraffin-embedded tissue blocks were stored at room temperature for more than 3 months. Total RNA extracted from epithelial cells microdissected from Carnoy's-fixed tissue samples contained intact template for up to a 977-base pair (bp) amplicon for beta-actin. Because of the emerging role of gene expression analyses in research, and in clinical work in the near future, an RNA-preserving fixative should replace formalin as the primary human tissue fixative. According to our data, Carnoy's fixative is an excellent candidate for a new primary fixing reagent for human tissue samples.     

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

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

Ethical implications of extracorporeal interval support for organ retrieval (EISOR)

With a growing demand for whole organs for transplantation, non-heart-beating organ donation is being revisited as a method of organ procurement. Extracorporeal interval support for organ retrieval (EISOR) is a new method to improve organ viability in non-heart-beating organ donation, but it potentially introduces ethical consequences.Our methodology included literature searches (Medline, Ovid, and bibliographies), abstract reviews, meeting presentations, and interviews with leaders in the field of organ donation and EISOR. No published articles on EISOR were found. Of the people interviewed (three EISOR enthusiasts, a medical ethicist, a Jewish rabbi, and a Catholic theologian), most agreed that premortem administration of systemic heparin was acceptable. Those who dissented stated that the potential of heparin to hasten a donor's death was unacceptable. All except one undecided person consented that the prevention of reanimation of the heart postmortem is an acceptable practice.With the advent of EISOR, the ethical issues surrounding the practice of premortem interventions and medications require more discussion among physicians, medical staff, and ethicists. Specifically, the dignity of death and premortem and postmortem interventions are discussed in this paper.     

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.     

Differential effects of transforming growth factor-beta(s) and glial cell line-derived neurotrophic factor on gene expression of presenilin-1 in human post-mitotic neurons and astrocytes.

Mutations in the presenilin-1 gene are linked to the majority of early-onset familial Alzheimer's disease cases. We have previously shown that the expression of transforming growth factor-beta is altered in Alzheimer's patients, compared to controls. Here we examine presenilin- expression in human post-mitotic neurons (hNT cells), normal human astrocytes, and human brain tumor cell lines following treatment with three isoforms of transforming growth factor-beta, or glial cell line-derived neurotrophic factor, a member of the transforming growth factor-beta superfamily. As the NT2/D1 teratocarcinoma cell line is treated with retinoic acid to induce differentiation to hNT cells, presenilin-1 messenger RNA expression is dramatically increased. Furthermore, there is a 2-3-fold increase in presenilin-1 messenger RNA expression following treatment of hNT cells with growth factors and similar results are found by Western blotting and with immunohistochemical staining for presenilin-1 protein. However, treatment of normal human astrocytes with cytokines results in minimal changes in presenilin-1 messenger RNA and protein. Interestingly, the expression of presenilin-1 in human U87 MG astrocytoma and human SK-N-SH neuroblastoma cells is only increased when cells are treated with glial cell line-derived neurotrophic factor or transforming growth factor-beta3. These findings suggest that endogenous presenilin-1 gene expression in human neurons can be induced by growth factors present in normal and diseased brain tissue. Cytokines may play a major role in regulating expression of presenilin-1 which may affect its biological actions in physiological and pathological conditions.     

Opportunistic infections in adults with acquired immunodeficiency syndrome: a comparison of clinical and autopsy findings.

BACKGROUND AND PURPOSE: Many opportunistic infections causing death in acquired immunodeficiency syndrome (AIDS) patients are often not diagnosed prior to death. The objective of this study was to compare the premortem and postmortem diagnoses of opportunistic infections and tumors among 15 AIDS patients treated in a hospital in southern Taiwan. METHODS: Total autopsy (brain, chest and abdominal cavity) was performed in 2 patients, and partial autopsy in 13. RESULTS: Pneumocystis carinii pneumonia, candidiasis, lymphoma, Kaposi's sarcoma, toxoplasmosis and salmonellosis were more commonly diagnosed before death than at autopsy. By contrast, cytomegalovirus (CMV) infections and herpes simplex virus or varicella-zoster virus infections were more frequently diagnosed at postmortem examinations than prior to death. CONCLUSIONS: In conclusion, this study found substantial discrepancies between autopsy findings and premortem clinical diagnoses in AIDS patients, especially for CMV infection.     

Body temperature is elevated in the early postmortem period

During the collection of specimens at autopsy for biochemical analysis, it was observed that body temperatures were higher than expected. To evaluate this observation further, rectal temperatures were determined at the earliest feasible time after death in 20 adult patients for whom recent premortem rectal temperatures were available for comparison. The average premortem temperature was 37.6 degrees C (range, 34.9 to 41.1 degrees C) and had been obtained 19 to 240 minutes (average, 107 minutes) before death. The average postmortem temperature, also 37.6 degrees C (range, 35.5 to 41.3 degrees C), was obtained 116 to 401 minutes (average, 202 minutes) after death. In the 11 patients in whom the postmortem interval was less than three hours (average, 155 minutes), there was an average postmortem temperature increase of 0.5 degree C (range, +1.3 to -0.7 degree C). The results suggest that there is usually an initial postmortem elevation in body temperature as measured rectally, probably as a result of continuing tissue and bacterial metabolism in the absence of the usual heat-dispersal mechanisms. This phenomenon should be considered when postmortem materials are used for analysis or when postmortem interval is determined by body temperature.     

High quality RNA retrieved from samples obtained by using LMPC (laser microdissection and pressure catapulting) technology

Isolation of intact RNA in high quality is the first and often the most critical step in performing many fundamental molecular biology experiments, and is essential for many techniques used in gene expression analysis. As many factors influence nucleic acid preservation, RNA isolation should include some important steps before and after the actual RNA extraction. We tested the influence of fixation and staining protocols regarding RNA integrity and concentration. A factor that is often underestimated is the absolute necessity for homogenous starting materials. Application of the LMPC technology allows for a rapidand highly precise procurement of purified cell populations suitable for a variety of downstream analyses.     

Medical autopsy: whose gain is it? An audit

Medical audit is essential in assessing the efficacy of health care delivery system. Though autopsy services are generally looked upon indifferently and with sceptism by the clinicians, it can form an important part of the medical audit system. The aims of this study were to audit autopsies of deaths within 24 hours of hospital admission by: 1) Comparing premortem and postmortem diagnosis; 2) Comparing postmortem gross diagnosis with postmortem histopathologic diagnosis; 3) Whether deaths could be certified based on clinical judgement and autopsies avoided. The study sample was 99 autopsies. In 45% autopsies, clinical impression did not match the final cause of death. In 14.2% autopsies, final cause of death could have been given by the clinician based on his clinical judgement. In 54.5% autopsies, there was agreement between premortem and postmortem diagnosis. In 67.6% autopsies, gross findings matched with the histopathologic findings.     

组织固定对核酸含量及完整性的影响

归档组织是回顾性研究丰富而巨大的资源库，但是往往因为组织的处理不当或者固定剂的影响使得组织中核酸含量严重降解，造成一些研究受到阻碍。如何保证组织中高质量核酸，重视组织的获得、处理、合适的固定剂及固定条件，是研究成功的前提条件。     

Varicella-Zoster virus gene expression at variable periods following death in a rat model of ganglionic infection

We used a rat model of Varicella-Zoster virus (VZV) ganglionic infection, which mirrors some of the features of VZV latency in humans, to determine the temporal pattern of expression of a VZV immediate-early gene (63) and a VZV late gene (40) at 0, 24 and 48 h after death of the animal. The immediate-early VZV gene 63 is known to be abundantly expressed during human ganglionic latency, while the late VZV gene 40 is not expressed during human latency. Using both RNA in situ hybridisation (ISH) and nested RT-PCR, it was found that at all time points in both thoracic and lumbar ganglia, the number of ganglia positive for VZV gene 63 was higher than for gene 40. The expression of gene 40 did not increase with time postmortem (pm) These results provide indirect support for the hypothesis that patterns of expression of VZV genes detected in human tissue at even 48 h pm reflect the pattern of expression during human ganglionic latency.     

RNA quality and gene expression analysis of ovarian tumor tissue undergoing repeated thaw-freezing

Gene expression profiles evaluated by microarray-based quantization of RNA are used in studies of differential diagnosis and prognosis in cancer. RNA of good quality is mandatory for this evaluation. The RNA most often comes from tumor banks with limited amount of tissue, and the tissue often undergoes repeated thawing and freezing. We evaluated the influence of repeated division of tumor samples at room temperature, on RNA quality and quantity, in addition to the gene expression profile. Sixteen ovarian tumor samples were divided in three aliquots each, undergoing respectively one, two, and three thaw-freeze cycles. RNA from each aliquot was extracted on the day of division, and quantity and quality were evaluated. RNA from all three aliquots of four tumor samples underwent microarray analysis on Affymetrix Human Genome U133A 2.0 arrays. Microarray data were evaluated using both unsupervised, and supervised multivariate statistical methods, reliability analysis, as well as verification using published gene lists in ovarian cancer studies. RNA quality and quantity did not change during the division procedure and microarray data showed insignificant difference in gene expression. Tumor samples from tumor banks can be frozen and thawed at least three times without compromising the RNA integrity and genetic expression profile.