A method to extract intact and pure RNA from mycobacteria

We describe a high-yielding, simple, and aerosol-free protocol for the isolation of RNA from mycobacteria that does not require sophisticated instruments. The method yielded 50 μg of RNA from 10⁷ cells, 50 times more than a recently reported method. Our method can extract total RNA from aerobically grown bacteria and from in vitro hypoxia-induced dormant bacilli and mycobacteria residing within infected macrophages.     

An improved method for isolation of RNA from rat femur

Background: RNA isolation from ossified bone is a difficult and time-consuming process which often results in poor recovery of RNA. The yield is limited and might not be suitable for gene quantification studies by real time PCR. Methodology: The present study demonstrates RNA extraction from rat femur utilizing the silica column along with the trizol reagent. Quality of RNA was assessed by agarose gel analysis and its suitability for real-time PCR analysis was determined by β-actin Ct values. Results: The RNA isolated using silica columns in conjugation with trizol reagent resulted in higher yield of RNA and purity (A260/280=2.04; yield =1545.73 µg/ml) compared to the trizol method alone (A260/280=1.85; yield =571.2 µg/ml). Ct value of β actin obtained from RNA isolated by trizol method was higher than the Ct value obtained by trizol in conjugation with the column method (31.41 and 15.41 respectively). Conclusion: Combination of trizol along with silica column resulted in better quality and improved yield of RNA suitable for gene quantification by Real time PCR.     

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.     

Effect of glucagon antiserum on somatostatin,glucagon and insulin release from the isolated perfused rat pancreas

In order to elucidate the effect of glucagon antiserum on the endocrine pancreas, the release of somatostatin, glucagon, and insulin from the isolated perfused rat pancreas was studied following the infusion of arginine both with and without pretreatment by glucagon antiserum. Various concentrations of arginine in the presence of 5.5 mM glucose stimulated both somatostatin and glucagon secretion. However, the responses of somatostatin and glucagon were different at different doses of arginine. The infusion of glucagon antiserum strongly stimulated basal secretion in the perfusate total glucagon (free + antibody bound glucagon) and also enhanced its response to arginine, but free glucagon was undetectable in the perfusate during the infusion. On the other hand, the glucagon antiserum had no significant effect on either insulin or somatostatin secretion. Moreover, electron microscopic study revealed degrannulation and vacuolization in the cytoplasm of the A cells after exposure to glucagon antiserum, suggesting a hypersecretion of glucagon, but no significant change was found in the B cells or the D cells. We conclude that in a single pass perfusion system glucagon antiserum does not affect somatostatin or insulin secretion, although it enhances glucagon secretion.     

石斛总RNA提取方法的研究

采用TRIZOL法、异硫氰酸胍法、Tris-硼酸法和改良的RNA提取方法提取石斛的总RNA,并通过凝胶电泳、紫外分光光度法检测提取的RNA样品的品质。研究结果表明:改良的RNA提取方法提取的RNA具有28S rRNA和18S rRNA两条清晰的条带,且无降解。OD260nm/OD280nm接近2.0,具有较高的纯度。其它三种方法获得的RNA品质较差,有降解和弥散现象。将改良的RNA提取方法提取的RNA逆转录成cDNA,经RAPD扩增,出现清晰的条带,进一步证明改良的RNA提取方法提取的RNA具有很高的纯度,可以满足进一步分子生物学研究的要求。     

The role of magnesium in regulating CCK-8-evoked secretory responses in the exocrine rat pancreas

This study investigates the effect of magnesium (Mg²⁺) on the secretory responses and the mobilization of calcium (Ca²⁺) and Mg²⁺ evoked by cholecystokinin-octapeptide (CCK-8) in the exocrine rat pancreas. In the isolated intact perfused pancreas CCK-8 (10^–10 M) produced marked increases in juice flow and total protein output in zero and normal (1.1 mM) extracellular Mg²⁺ [Mg²⁺]_o compared to a much reduced secretory response in elevated (5 mM and 10 mM) [Mg²⁺]_o Similar effects of perturbation of [Mg²⁺]_o on amylase secretion and ⁴⁵Ca²⁺ uptake (influx) were obtained in isolated pancreatic segments. In pancreatic acinar cells loaded with the fluorescent bioprobe fura-2 acetomethylester (AM), CCK-8 evoked marked increases in cytosolic free Ca²⁺ concentration [Ca²⁺]_i in zero and normal [Mg²⁺]_o compared to a much reduced response in elevated [Mg²⁺]_o Pretreatment of acinar cells with either dibutyryl cyclic AMP (DB₂ cAMP) or forskolin had no effect on the CCK-8 induced changes in [Ca²⁺]_i. In magfura-2-loaded acinar cells CCK-8 (10^–8 M) stimulated an initial transient rise in intracellular free Mg²⁺ concentration [Mg²⁺]_i followed by a more prolonged and sustained decrease. This response was abolished when sodium Na⁺ was replaced with N-methyl-D-glucamine (NMDG). Incubation of acinar cells with 10 mM Mg²⁺ resulted in an elevation in [Mg²⁺]_i. Upon stimulation with CCK-8, [Mg²⁺]_i. decreased only slightly compared with the response obtained in normal [Mg²⁺]_o. CCK-8 caused a net efflux of Mg²⁺ in pancreatic segments; this effect was abolished when extracellular sodium [Na⁺]_o was replaced with either NMDG or choline. The results indicate that Mg²⁺ can regulate CCK-8-evoked secretory responses in the exocrine pancreas possibly via Ca²⁺ mobilization. Moreover, the movement of Mg²⁺ in pancreatic acinar cells is dependent upon extracellular Na⁺.     

An optimized freeze-squeeze method for the recovery of DNA fragments from agarose gels

A procedure for quick and simple elution of DNA from agarose gels is presented. After electrophoresis, bands of interest are cut out of the gel and the slices are equilibrated in a neutral salt buffer. The slices are then frozen and centrifuged through a filtration assembly whereby the DNA-containing buffer is squeezed out. The method is simple, quick, and suitable for the safe handling of small amounts of DNA (less than 1 microgram). The isolated DNA is susceptible to any enzymatic reaction and also to chemical sequencing. The method is most useful for rapid preparation of specifically end-labeled DNA fragments (e.g., for sequencing), but may also be utilized for any other preparative applications.     

Extraction of RNA from Ca-alginate-encapsulated yeast for transcriptional profiling

We have developed a method for preparing high-quality total RNA from Ca-alginate-encapsulated Saccharomyces cerevisiae that is suitable for microarray analysis. Encapsulated cells were harvested from immobilized cell reactors and flash-frozen in liquid nitrogen. Following low-temperature mechanical disruption, cells were freed from Ca-alginate by reverse ionotropic gelation and purified by centrifugation, and then total RNA was extracted using hot acid phenol. The yield and quality of the RNA were consistently high; the RNA was free of contaminating alginate, and in microarray analysis it performed as well as RNA isolated from planktonic cells.     

小麦总RNA的快速提取

Rneasy Kits用于从小量小麦中分离总RNA。它为同时和多次制备各种生物样品提供了一种快捷而简便的方法-整个过程可以在30分钟内完成。而且Rneasy方法中不再涉及使用有毒物质,如酚类、氯仿等。通过此方法纯化的RNA可用于各种标准的下游技术,如RT-PCR、polyA^ RNA选择、差异显示技术、Northern点杂交和狭线杂交、引物延伸、cDNA合成、陈列表达分析和表达芯片分析等。使用这个盒子,我们多次成功的进行了小麦总RNA的分离,其中的三次在本文中进行了分析。OD260nm/OD280nm介于1.7-2.0之间,OD260nm/OD230nm大于2.0.说明RNA纯度很高,未被蛋白质、苯酚等物质污染。     

黄檗（Phellodendron amuranse）叶片总RNA提取方法研究

以黄檗(Phellodendron amuranse Rupr.)叶片为材料,分别利用改进的盐酸胍法、Trizol法、CTAB法提取黄檗叶片总RNA,通过RNA产率、纯度、电泳图谱等分析,确立了1种从黄檗叶片中快速分离总RNA的方法。研究结果表明,改进盐酸胍法所提取的总RNA的A₂₆₀/A₂₈₀为1.928,28S和18S条带清晰谱图完整性好,而且具有产率高、时间短、成本低的特点,所提取的总RNA适用于mRNA分离、cDNA文库的建立、Northern杂交等分析。     

一种适于提取荔枝花与幼果组织总RNA的方法

介绍了一种从荔枝花与幼果组织中提取高质量和较高产量的总RNA的方法,该方法提取的总RNA可以满足构建cDNA文库、开展RT-PCR、Northern杂交分析、基因表达差示分析等方面研究的要求。     

从血液中提取总RNA的一种快速高效方法

血液中含有大量的RNA酶 ,可引起RNA的降解 .防止RNA酶的降解 ,是保证所得RNA片段完整的关键 .目前提取RNA的方法较多 ,但有些方法尚不能完全防止RNA降解 .将TRIZOL方法稍加改进 ,将TRIZOL与异硫氰酸胍联用提取血液淋巴细胞总RNA .琼脂糖凝胶电泳结果表明 ,其 2 8SRNA与 18SRNA的比值为 2∶1,优于单独使用其中任何一种试剂者 .此方法同样适用于从其它细胞中提取RNA .     

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。     

多花蔷薇总RNA提取方法

根据总RNA完整性、纯度和得率筛选出适合多花蔷薇幼嫩根、叶总RNA的提取方法。结果表明,以CTAB/酸酚法提取的扦插苗根系总RNA、以LiCl-尿素法提取的扦插苗根、叶总RNA以及采用RNeasyPlantMiniKit试剂盒的改进方法提取的组培苗嫩叶总RNA电泳有清晰明亮的28S、18S条带,无降解;其A260/A280值为1.73~2.04,表明总RNA质量好。RT-PCR结果进一步证实所提取的总RNA能够用于分子生物学的各种下游实验。RNA得率分别为:根系和组培苗嫩叶120-140μg/g(fw),扦插苗嫩叶190-230μg/g(fw)。CTAB/酸酚法提取的嫩叶总RNA、SDS/酸酚法提取的根、叶总RNA有多糖污染,且有明显降解。TotalRNAisolationsystem(Z5111,Promega)试剂盒不适合提取多花蔷薇各组织总RNA。     

Isolation of RNA from mycobacteria grown under in vitro and in vivo conditions

Isolation of RNA from mycobacteria is very difficult to perform, and the yields are generally very low. We describe an approach to isolate RNA from mycobacterial species which combines the disruption of mycobacterial cells by a silica/ceramic matrix in a reciprocal shaker with the ease and efficiency of subsequent RNA purification on spin columns with silica gel-based membranes. This method is rapid, easy to perform and yields high amounts of pure, intact total RNA. Due to its safety, this method is applicable even to group 3 biological hazard organisms like Mycobacterium tuberculosis. By combining a method for the isolation of phagosomal bacteria from infected primary macrophages with the novel RNA isolation technique, we are able to monitor gene expression during infection even in bacteria which are rather resistant to genetic manipulation, like Mycobacterium bovis.     

Method for the isolation of high-quality RNA from grape berry tissues without contaminating tannins or carbohydrates

Grape berries contain compounds that aggregate with and precipitate RNA in the presence of chaotropic agents or phenol. The procedure described here extracts RNA from finely ground tissues using mild denaturants, and selectively precipitates the aggregate-forming material with 30% ethanol. The resulting RNA is suitable for northern blot analysis and translationin vitro.     

Using a commercial DNA extraction kit to obtain RNA for RT-PCR from starchy rice endosperm

The extraction of RNA from a starchy plant material, such as many common food grains, is difficult, and especially so from the mature endosperm of rice. Most commercial RNA kits are not suitable for starchy materials. Traditional RNA extraction procedures, in addition to being laborious and time consuming, leave hazardous organic wastes that result in expensive disposal costs. Interestingly, the numerous commercial DNA isolation kits now available often include directions for eliminating co-isolated RNA. This indicated an approach to obtain the generally unwanted RNA by-product by treating the total extraction product to intentionally retain RNA. A method was developed by which a two-step DNase procedure was applied to the product of the Cartagen Food DNA extraction kit that eliminated the DNA but left the co-extracted RNA. This modified procedure was compared with several other commercial and standard methods that are promoted as being able to work under high polysaccharide conditions. Successful extraction was determined by the production and amplification of cDNA by RT-PCR of actin. Extraction was successful from milled rice, as well as from cornmeal and wheat flour. The modification provides an RNA extraction method that is quick, easy, and inexpensive, and also eliminates the production of hazardous wastes.     

一种高效提取棉花RNA的改良方法

与其他植物相比,棉花组织中含有较多的酚类、萜类和多糖等次生代谢物,这些物质严重影响棉花细胞中RNA的分离。针对棉花富含次生代谢物的特点,同时简化操作步骤,摸索出一种高效提取棉花RNA的方法,该方法具有提取RNA完整性好、纯度高和操作简单等特点,可适用于提取棉花等多糖多酚类植物的RNA。