鼠抗人转铁蛋白抗体轻链与重链可变区基因库的制备

以纯化的人转铁蛋白免疫小鼠,测其血清效价,合乎要求后杀鼠取脾脏并以异硫氰酸胍和酚的混合溶液将其匀浆,再以氯仿抽提、异丙醇沉淀和乙醇洗涤,所得总RNA以oligo(dT)纤维素离心柱分离纯化制备mRNA,使mRNA达到eDNA合成所需的浓度后,反转录制备出eDNA,在合成的eDNA中加入轻、重链引物,扩增制备出小鼠抗人转铁蛋白抗体的轻、重链可变区基因库。     

不同死因大鼠心肌内多种RNA相对表达量变化与PMI的关系

目的观察不同死因下大鼠心肌内多种RNA相对表达量随死亡时间（PMI）的变化。方法建立断颈死、窒息死和失血性休克死大鼠模型,提取心肌中总RNA,利用实时荧光定量PCR技术检测甘油醛-3-磷酸脱氢酶（glyceraldehyde～3-phosphatedehydrogenase,GAPDH）,β-肌动蛋白（β-actin）、诱导型-氧化氮合酶（induciblenitricoxidesynthase。iNOS）、缺氧诱导因子-1（hypoxia-induciblefactor-1,HIF-1）、肿瘤坏死因子-d（tumornecrosisfactor-d,TNF-a）、白细胞介素-6（interleukin-6,IL-6）tuRNA以及U6微核RNAfU6sreallnuclearRNA。U6snRNA）的循环阈值,观察各指标相对表达量随PMI的变化情况。结果U6snRNA表达稳定,可作为内对照。在死亡早期,CAPDH、HIF-1、iNOS、TNF-d和IL-6的相对表达量在窒息组和失血性休克组比断颈组呈现不同程度的增加,而β-actin在3个组皆呈现相对表达量下降趋势。在死亡晚期,随着RNA的不断降解,相对表达量持续下降。结论死后各RNA相对表达量的特征性变化可为不同死因下的PMI推断提供-定的参考。     

现场生物斑迹遗留时间推断研究进展

就现场提取的生物检材而言,我们除了需要准确获得DNA STR分型进行个体识别和亲子鉴定,还希望能够通过技术手段获知其遗留时间、空间定位等更多信息。本文以血迹为主要研究对象,就国内外采用光学、细胞生物学、分子生物学等方法推断生物斑迹遗留时间进行总结,并对法医微生物学这一法庭科学新领域在时空线索推断中的应用做以介绍,为现场生物斑迹遗留时间推断研究提供参考。     

The development of a method for FISH identification of forensically relevant body fluids

Messenger RNA profiling is a useful confirmatory test for body fluid identification, but there are limitations to this method including sensitivity and the difficulty in linking body fluids with the corresponding DNA profile in mixed samples. We have developed a method for RNA suspension fluorescence in situ hybridisation (RNA S-FISH) for forensic-type samples using locked nucleic acid (LNA) probes. Vaginal and buccal epithelial cells have been the primary focus, with some preliminary work performed on leucocytes and seminal round cells. We have designed probes for the KRT10 messenger RNA and the micro RNA 891a. Using these probes, we have optimised a RNA S-FISH methodology and have successfully visualised these cell types using fluorescent microscopy.     

核酸与死亡时间的相关性及其检测方法

准确推断死亡时间一直是法医学研究的热点和难点。躯体死后组织细胞自溶,核酸（DNA和RNA）降解,在一定时间内其含量呈下降趋势并与死亡时间有线性关系。文章就核酸降解与死亡时间的相关性及核酸含量的检测方法进行综述,以获得对该技术的全面了解,并对解决死亡时间推断这一法医学难题的应用前景进行初步探讨。     

不同温度下大鼠皮肤5种RNA指标与PMI的相关性

目的探讨大鼠皮肤内β-肌动蛋白(β-actin)、甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)、18S核糖体RNA(18S ribosomal RNA,18S rRNA)、5S核糖体RNA(5S ribosomal RNA,5S rRNA)以及微小RNA-203(microRNA-203,miR-203)这5种RNA指标在不同温度下的表达水平与死亡时间(PMI)的相关性。方法将18只SD大鼠随机分为3组,处死后分别置于4℃、15℃和35℃的环境中,于死后0~120h内11个时间点取大鼠腹部皮肤。抽提皮肤总RNA,利用实时荧光定量PCR技术检测5种RNA表达水平。运用geNorm软件选取合适内参后,利用GraphPad软件对内参标准化的RNA指标进行回归分析。结果 5S rRNA和miR-203作为内参最合适。在4℃和15℃温度组中,β-actin和GAPDH的表达量变化与PMI线性关系良好。在35℃下,β-actin和GAPDH与PMI呈现S形曲线关系。而18S rRNA只在15℃和35℃温度组呈现一定的线性关系。结论皮肤组织比较适合作为提取RNA的检材,其中β-actin和GAPDH表达水平与PMI相关性良好,有望成为推测PMI的辅助指标。     

Gene expression during blow fly development: improving the precision of age estimates in forensic entomology

Forensic entomologists use size and developmental stage to estimate blow fly age, and from those, a postmortem interval. Since such estimates are generally accurate but often lack precision, particularly in the older developmental stages, alternative aging methods would be advantageous. Presented here is a means of incorporating developmentally regulated gene expression levels into traditional stage and size data, with a goal of more precisely estimating developmental age of immature Lucilia sericata. Generalized additive models of development showed improved statistical support compared to models that did not include gene expression data, resulting in an increase in estimate precision, especially for postfeeding third instars and pupae. The models were then used to make blind estimates of development for 86 immature L. sericata raised on rat carcasses. Overall, inclusion of gene expression data resulted in increased precision in aging blow flies.