Improved method for the isolation of RNA from bacteria refractory to disruption, including S. aureus producing biofilm

The development of fast, reliable and inexpensive phenol protocol is described for the isolation of RNA from bacterial biofilm producers. The method was tested on Staphylococcus aureus (S. aureus) and other biofilm-producing gram-negative microorganisms and provided the highest integrity of RNA recovery in comparison to other methods reported here. In parallel experiments, bacterial lysis with Qiagen, NucleoSpin RNAII, InnuREP RNA Mini, Trizol and MasterPure RNA extraction Kits using standard protocols consistently gave low RNA yields with an absence of integrity. The boiling method presented here yielded high concentration of RNA that was free from 16S and 23S rRNA, contained 5S RNA. Higher yields due to improved biofilm bacterial cell lysis were achieved with an added hot phenol incubation step without the need for a bead mill or the enzyme. This method when used in conjunction with the Qiagen RNeasy Mini kit, RNA isolation was a success with greater integrity and contained undegraded 16S and 23S rRNA and did not require further purification. Contaminating DNA was a problem with the RNA processing samples; we used quantitative real-time PCR (RT-qPCR) to measure the recovery of RNA from bacterial biofilm cells using the method described here.     

Preparation of DNA and four different RNA species from rat brain. A new RNA fraction and a new characteristic of the various RNA's

A new modification is described for the fractionation of nucleic acids from brain. The cold phenol method followed by a hot extraction was combined with ‘salt-precipitation’ and chromatography on Sephadex and Agarose gels. The method enables the isolation of five highly purified fractions in large amounts: DNA; high molecular weight salt-soluble RNA, a hitherto undescribed fraction; rRNA; residual RNA (mRNA) and sRNA. All fractions showed, after 9 h in vivo incubation with ³²P, different specific radioactivities and/or characteristic base compositions. The single mononucleotides of each RNA fraction differed markedly in their ³²P-labelling rate. This was found to be a new characteristic, for these differences were in no way similar in the various RNAs, but specific and constant for each fraction. An assay of base composition only by ³²P-labelling was therefore not possible.     

快速提取棉花蕾期高质量RNA的改良方法

棉花组织中因富含棉酚、多糖和单宁等次生代谢物,RNA难分离,易降解,导致现有RNA快速提取试剂盒提取的棉花RNA产率低、质量差,无法应用于实验操作。针对棉花组织次生代谢物多的特点,以酸酚法为基础,结合RNA吸附柱,通过对幼苗期和蕾期棉花叶片RNA提取,总结出一种快速提取棉花组织RNA的方法。与热硼酸法、酸酚法及试剂盒法相比,所述方法具有步骤少、产率高、质量好等特点,操作简单、整个实验在1h内完成,可开发出提取多糖多酚类生物组织RNA试剂盒。     

自然干燥紫萼藓总RNA提取方法

介绍一种适合富含酚类、萜类等次生物质的干燥紫萼藓的总RNA的提取方法—SDS/酸酚法。采用SDS做为去污剂,用水饱和酚、氯仿和异戊醇进行抽提以去除蛋白、酚类等次生物质,醋酸钾和无水乙醇去除多糖等物质,最后LiCl沉淀获得总RNA。该方法不但获得了完整性好和纯度高的RNA,而且操作简单,成本也较低,对其他富含酚类、萜类等次生物质的干燥植物组织的总RNA的提取具有借鉴意义。     

一种高效经济的高质量植物RNA提取方法

建立了一种高效经济的植物RNA提取方法.在提取缓冲液中加入蔗糖、氯化钾和镁离子以提供对RNA分子的保护.破碎后的细胞于提取缓冲液中裂解后,用酚/氯仿变性并去除内源RNA酶和其他蛋白质,而后用pH 5.6 的NaAc沉淀RNA.用该方法提取RNA的得率较高,经电泳检测,RNA的完整性很好.RNA印迹分析和RT-PCR也都得到很好的结果.该方法还使实验成本大大降低.     

Direct extraction and purification of rRNA for ecological studies.

Microgram quantities of rRNA were recovered from natural microbial communities in sediment, soil, and water with a lysozyme-hot phenol direct extraction method. Gel filtration with Sephadex G-75 spun columns readily removed humic-like contaminants without any measurable loss of rRNA and rendered RNA extracts of sufficient quality for molecular procedures.     

A method for isolation of RNA from Pneumocystis carinii

Total RNA from Pneumocystis carinii obtained directly from the rat lung and from short term culture on A549 cells was evaluated for size and purity. An isolation procedure using guanidine isothiocyanate and lithium chloride was preferable to a hot phenol method. Host cells were eliminated by hypotonic lysis and a series of microfiltrations. Pneumocystis carinii were pretreated with Zymolyase for increased susceptibility to chaotropic agents. The major ribosomal species of P. carinii RNA migrated similarly to Saccharomyces cerevisiae rRNA. The 28s-like species migrated well ahead of rat and A549 cell rRNA and well behind the prokaryotic large rRNA species.     

A Method for Isolation of RNA from Pneumocystis carinii

Toial RNA from Pneumocystis carinii obtained directly from the rat lung and from short term culture on A549 cells was evaluated for size and purity. An isolation procedure using guanidine isothiocyanate and lithium chloride was preferable to a hot phenol method. Host cells were eliminated by hypotonic lysis and a series of microfiltrations. Pneumocystis carinii were pretreated with Zymolyase for increased susceptibility to chaotropic agents. The major ribosomal species of P. carinii RNA migrated similarly to Saccharomyces cerevisiae rRNA. The 28s-like species migrated well ahead of rat and A549 cell rRNA and weli behind the prokaryotic large rRNA species.     

一种从富含多糖的玉米幼穗中提取RNA的方法

介绍了一种大量提取玉米(Zeamays L.)幼穗总RNA的有效方法。由于玉米幼穗富含多糖,用普通的RNA提取方法和Trizol很难获得高质量的RNA。在热酚法提取RNA的基础上,通过在提取缓冲液中加入低pH值的醋酸钾来去除多糖。所得到的RNA获得率高,质量好,条带完整,实验证明可直接用于RT-PCR、微阵列等各项后续分子生物学实验。     

一种从富含多糖的玉米幼穗中提取RNA的方法

介绍了一种大量提取玉米(Zea mays L.)幼穗总RNA的有效方法。由于玉米幼穗富含多糖, 用普通的RNA提取方法和Trizol很难获得高质量的RNA。在热酚法提取RNA的基础上, 通过在提取缓冲液中加入低pH值的醋酸钾来去除多糖。所得到 的RNA获得率高, 质量好, 条带完整, 实验证明可直接用于RT-PCR、微阵列等各项后续分子生物学实验。     

一种提取高质量油菜种子和种皮RNA的方法

提取高质量的RNA是从基因表达水平上研究油菜种子和种皮发育的必要条件。现有方法因为油菜种子脂肪、多酚和多糖,难以快速获得完整、高纯度的油菜种子总RNA。本试验针对油菜种子和种皮特点,利用苯酚-氯仿抽提后用无水乙醇沉淀RNA,建立了在油菜种子和种皮中快速提取高质量总RNA的提取方法,电泳分析表明28S rRNA亮度约为18S rRNA的2倍;紫外分光光度计检测A260/A280介于1.8～2.0之间。用该法分离的RNA,已成功用于RT-PCR、Northern blot分析和基因全长的克隆等分子生物学研究。