生物样本库中血液样本microRNA和DNA的检测方法学探讨

目的探讨建立南京医科大学第一附属医院生物样本库中血液生物样本microRNA的检测方法以及血凝块中DNA的鉴定方法。方法随机选取本院生物样本库自2012-2014年存储的胃癌、肝癌血浆标本进行microRNA的提取,后经Q-PCR验证miR-21和miR-387的表达情况以及将对应的血凝块样本进行全血样本DNA的提取,通过全自动核酸分析仪鉴定提取的DNA的完整性。结果 Q-PCR结果表明,冻存血浆在保存了1、2、3年后都能检测到相关的microRNA的表达,同时血凝块中提取的DNA,经全自动核酸分析仪鉴定,DNA完整性也较好。结论该方法能够初步验证我院生物样本库中冻存的血样标本中的RNA和DNA的保存质量,血样质量基本能满足后续科研实验的需求。本研究中的血样标本质量鉴定的方法切实、可行,能较准确地反映生物样本库中血样本的质量,为今后我院组织样本的质量控制体系的建立奠定基础。     

血液放置时间和RNA反复冻融对RNA质量的影响

正在临床和科研工作中,血液和组织是比较容易获取的生物标本,特别适用于临床和实验室检测及生物样本库收集保存。DNA、RNA和蛋白质是三种重要的生物大分子,其中RNA在人类多种生理和病理过程中发挥着举足轻重的作用。RNA通常是从组织、全血和培养的细胞中提取获得~([1]),高质量RNA可满足RT-PCR、Northern blot、基因表达谱、转录组测序和阵列分析等生物学实验的研究需求~([2-3])。由于     

朊病毒病实验动物脑组织PrPSc样本库的建立

目的 建立朊病毒病实验动物脑组织检测样本库,检测其在特定保存条件下的稳定性,以用于动物和人朊病毒病诊断技术的评估和考核.方法 选用30只经颅内注射感染羊瘙痒因子263K的仓鼠和30只正常仓鼠,每只分别制备10%、1%和0.5%3种浓度的脑组织匀浆,分装冷冻保存.以Western Blot方法 确定脑组织匀浆中PrPSc的存在情况,并分别在建库半年和3年检测PrPSc的稳定性.结果 30只感染仓鼠10%脑组织匀浆全部可检出PrPSc,1%脑组织匀浆有26份为PrPSc阳性,0.5%脑组织匀浆有19份为PrPSc阳性.半年及3年后复检,PrPSc阳性检出率基本不变.所有正常仓鼠脑匀浆均为PrPSc阴性.结论 建立了稳定性良好的含有90份PrPSc阳性标本和90份PrPSc阴性样本的朊病毒病实验动物脑组织检测样本库.     

高血压项目临床检测样本保存和稳定性标准化研究

目的 探讨不同保存温度和时间条件下高血压五项检测血浆样本的稳定性.方法 收集20例Renin、ALD、ACTH、Cortisol水平不同的受试者样本,分别保存在不同温度(常温、4℃、-20℃、-80℃)和不同时间(24h和48h),另外20例AII水平不同的受试者样本,分别在不同时间(即时、2h、4h、8h、12h、24h)添加酶抑制剂,并在不同温度和时间(常温下2h、4h、6h、8h,4℃下4h、8h、12h、24h,-20℃下12h、24h、48h、72h)下保存,计算偏倚.结果 在室温保存条件下,Renin浓度在24h内降低9%,而4℃条件下保存,24h内升高14%,在-80℃保持稳定.ACTH浓度在室温保存下降低32%,-20℃和-80℃条件下保存比较稳定.ALD和Cortisol浓度受保存温度及时间变化影响较小,48h内变幅在5%以内.AII在采血后的不同时间段添加酶抑制剂,8h后升幅超过10%;样本采集后立即添加酶抑制剂,在常温保存8h条件下及4℃下保存24h后均减少超过50%,而在-20℃条件下48h降幅在10%以内.结论 Renin和ACTH需在-20℃以下的低温保存,ALD和Cortisol对温度没有要求,AII需立即加入酶抑制剂并尽快检测,如果不能尽快检测,需分离血浆后保存在-20℃条件下,并在48h之内完成检测.本研究为临床检验制定完善的高血压五项样本保存条件提供了依据.     

标本保存时间、温度对荧光定量HBV-DNA检测的影响

目的 分析乙型肝炎患者血清样本的保存时间和温度对乙型肝炎病毒DNA定量检测的影响.方法 选取2015年1月至2015年4月于满洲里市第一医院PCR实验室进行乙型肝炎病毒DNA定量检测的阳性患者血清,分别于室温和4℃条件下保存1d和7d,利用荧光定量PCR方法进行病毒定量检测.结果 相同保存时间下,室温和4℃保存的血清样本病毒DNA定量检测无统计学差异:相同保存温度下,1d和7d保存的血清样本病毒DNA定量检测无统计学差异.结论 采用荧光定量PCR方法检测乙型肝炎患者血清病毒DNA的含量,其结果在7d内不受样本保存时间和温度的影响.     

儿童专科医院特色生物样本库的建立与应用

<正>生物样本库是一种集中收集、处理、存储和应用各种生物材料及其相关信息用于疾病的临床诊疗研究的生物应用系统~([1])。一般由两部分组成:一是样本实体库,即可以从中提取到DNA、RNA、蛋白质等成分的组织或血液等生物样本;二是样本信息库,包括从生物样本中提取DNA、RNA、蛋白质等成分所得到的个人独有的遗传信息数据及临床诊疗信息~([2])。早在1994年,中国科学院就建立了中华民族永生细胞     

高效快速提取股骨头中总RNA的方法

背景：从骨组织中有效提取总RNA是进行骨科相关实验的基础,目前所知方法的提取效果不理想。 目的：探寻一种高效、快速的骨组织总RNA提取方法。 方法：取健康大耳白兔股骨头迅速置于用液氮预冷过的研钵中,液氮中反复研磨至粉末状,再将其移至预冷的匀浆器内,加入Trizol,充分匀浆后4 ℃离心,取上清,加入氯仿离心,使RNA与细胞DNA、蛋白质及其他成分分离从而得到总RNA。另取兔股骨头,按传统方法取材后保存,无匀浆过程,传统Trizol法提取总RNA作为对照。紫外分光光度计测定RNA样品浓度、纯度及产率。甲醛变性胶电泳观察RNA的28 S,18 S条带是否清晰,有无降解和DNA污染。 结果与结论：实验方法提取的总RNA浓度在0.80~0.90 g/L,A260/A280在1.90~2.00之间,A260/A230在1.4~1.6之间,明显高于传统方法提取的RNA各指标,说明实验方法提取的总RNA纯度高,无DNA、蛋白质污染。甲醛变性胶电泳可清晰显示28 S,18 S两条带,大体观察其比例大约为1∶1,证实RNA提取完整,没有降解。由此可见,实验方法提取的骨组织总RNA质量高,提取方法方便、快捷,可用于骨组织分子生物学研究。     

肺结核中DNA低甲基化趋势与TETs及TDG的相关性研究

目的明确肺结核病人DNA低甲基化状态与TETs、TDG之间的相关性。方法在前期研究结果的基础上,收集健康对照和活动性肺结核病人治疗前全血RNA,通过实时荧光定量PCR(Real-time PCR)方法,对参与DNA甲基化下调的DNA去甲基化酶,即十-十一染色体异位酶基因(TETs,包括TET1、TET2、TET3)及胸腺嘧啶糖基酶(TDG)进行定量检测。利用H37Rv裂解抗原以及5-氮杂胞嘧啶核苷(5azac)刺激物刺激人肺癌细胞系A549和人支气管上皮细胞Beas-2B两种细胞系,并收集刺激后不同时间点细胞DNA、RNA。利用甲基化敏感性限制性分析(MSRA)对所收集DNA样本进行分析;进一步利用Real-time PCR(RT-PCR)方法对细胞RNA样本进行检测。结果在肺结核病人组中,DNA去甲基化酶表达均显著升高(P0.05)。H37Rv抗原、5azac导致DNA甲基化水平降低,且刺激时间越长甲基化降低越明显。且在刺激第4天A549细胞中H37Rv刺激组DNA去甲基化酶TET2、TET3上调,而在Beas-2B中为H37Rv刺激组DNA去甲基化酶TDG上调,与临床检测结果相符。结论在活动性肺结核病人中及体外细胞刺激实验H37Rv抗原刺激组中,DNA甲基化呈现低甲基化趋势;DNA去甲基化酶可能是参与其DNA低甲基化趋势的主要的酶。     

继发性高血压相关检测项目不同条件下尿液标本的稳定性研究

目的 分析不同保存温度及不同保存时间继发性高血压相关生物标志物醛固酮(aldosterone, ALD)、17-羟类固醇(17-hydroxysteroid, 17-OHCS)、17-酮类固醇(17-keto steroids, 17-KS)、香草扁桃酸(vanilla mandelic acid, VMA)、微量白蛋白(microalbumin, MALB)及肌酐(creatinine, CREA)尿液样本的稳定性,为临床样本的前处理及检测提供实验依据。方法 收集健康志愿者新鲜尿液样本,利用标准品制备不同浓度尿液样本,高低浓度均匀分布,在加速37℃、室温(18～26℃)、冷藏(2～8℃)、冷冻(-10～-20℃)等不同条件下保存不同时间后分别进行检测,运用Bland-Altman法和偏差分析观察6项指标的一致性和偏差。结果 醛固酮、17-酮类固醇、肌酐尿液样本保存于37℃,可稳定放置11h; 17-羟类固醇尿液样本可稳定8h;香草扁桃酸和微量白蛋白尿液样本不宜在37℃放置。保存于18～26℃条件下,醛固酮尿液样本可稳定放置24h; 17-羟类固醇、香草扁桃酸和肌酐尿液样本可稳定放置4...     

Optimisation of postmortem tissue preservation and alternative protocol for serotonin transporter gene polymorphisms amplification in SIDS and SIUD cases

The major obstacle to genetic research in SIUD (sudden intrauterine unexplained death) and SIDS (sudden infant death syndrome) cases is the complex characteristics of the human anatomic samples available. In fact, in Italy autopsies are performed at least 24 h post-mortem and tissues can be left in formalin for long fixation times (> 4/5 days), thus compromising nucleic acids integrity. In this study we compared the quality of DNA and RNA extracted from tissues differently preserved. As expected, the DNA and RNA from formalin-fixed and paraffin-embedded tissues, formalin-acetic acid-alcohol tissues and ethanol tissues were of poor quality and not adequate for subsequent molecular analysis. The best results were obtained with RNAlater preserved tissues: this buffer was equivalent, if not superior, to freezing method for preservation of postmortem DNA and RNA.In addition, we introduce a new protocol for the amplification of the serotonin transporter gene promoter region (5-HTT) ideal to obtain the increase of specific product, avoiding artifacts formation.     

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.     

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.     

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.     

Time-dependent changes in post-mortem testis histopathology in the rat

To clarify the contribution of spontaneous or autolytic post-mortem changes to testis histopathology, the testes of adult rats were examined after animals were left at room temperature for 12, 24, 36, and 48 hours postmortem (n = 2 for all time points except 0 hours postmortem, where n = 3). A progressive decrease in testis weight and seminiferous tubule diameter was observed, as well as detachment of the seminiferous epithelium from the basement membrane. As early as 12 hours postmortem, there was observable clumping and margination of chromatin in Leydig cells, Sertoli cells, spermatogonia, spermatocytes, and step 7-10 spermatids; extensive disintegration of Sertoli cells and residual bodies by 24 hours postmortem; and TUNEL positivity of Leydig cells (by 36 hours postmortem) and step 19 spermatids (at 48 hours postmortem). These findings will aid in ensuring proficient histopathological analysis of testes in toxicity studies.     

Recombinant DNA studies on stored necropsy brain samples from patients with Huntington''s chorea.

An analysis of deoxyribonucleic acid (DNA) in deep frozen brain samples taken from 100 patients with Huntington's chorea after death showed undegraded DNA in 44 cases. Of these, 16 were analysed with G8, a recombinant DNA probe, linked to the Huntington's chorea locus. In all cases unambiguous Southern blots were obtainable. No correlation between the yield of DNA and the principal storage factors was observed. The use of stored brain tissue obtained after death from patients with Huntington's chorea should be considered when analysis of DNA is needed for predictive studies, but DNA should preferably be isolated from the tissue before storage as degradation in these samples can occur.     

8OHdG levels in brain do not indicate oxidative DNA damage in Alzheimer''s disease

Accumulation of oxidative DNA damage has been proposed to underlie aging and neurodegenerative diseases such as Alzheimer's Disease (AD). The DNA adduct 8-hydroxy-2′-deoxyguanosine (8OHdG) is considered a good indicator of oxidative DNA damage. To investigate whether this type of DNA damage is involved in AD etiology, 8OHdG levels were determined in postmortem human brain tissue of controls and AD patients (in frontal, occipital, and temporal cortex and in hippocampal tissue). Parametric studies in rat revealed no influences of postmortem delay, repeated freezing/thawing or storage time. In human brain, approximately two 8OHdG molecules were present per 10⁵ 2′-deoxyguanosines. In AD patients and controls, 8OHdG-levels were not related to age, sex, or brain region. Also, no differences were found between controls and AD patients. It was concluded that 8OHdG in nuclear DNA, although present throughout the brain in fairly high amounts, does not accumulate with age, nor does it appear to be involved in AD. More detailed studies are required to extend this conclusion to other types of oxidative damage.     

Quantitative temporal-spatial distribution of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) in post-mortem tissues

Few post-mortem studies have been performed on patients who have died from severe acute respiratory syndrome (SARS). No studies have examined how the SARS-associated coronavirus (SARS-CoV) loads in different organs with respect to time, post-mortem. The aim of this study was to determine the quantitative temporal-spatial distribution of SARS-CoV in the post-mortem tissue samples of seven patients. Quantitation of a house-keeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was undertaken to standardize the amount of tissue tested. SARS-CoV viral load and SARS-CoV/GAPDH RNA ratio for each organ type were related to four time durations: onset of illness to death, death to post-mortem tissue sampling, and total durations of treatment with ribavirin and hydrocortisone. The SARS-CoV/GAPDH RNA ratio remained relatively stable in most organ tissue types for all these time durations. The ratio reached the highest value of equal to or greater than one for lung and small bowel, whereas those for heart, liver, spleen, and kidney were always less than one. It is concluded that SARS-CoV viral loads in these organs remain relatively stable, post-mortem. This quantitative assessment further supports SARS-CoV has a specific tropism for the human respiratory and gastrointestinal tracts, which may be related to the density of SARS-CoV receptors.