Simultaneous Isolation Of RNA Research Articles

Comparison of RNA extraction methods from <i>Microcystis</i> colonies

微囊藻群体富含多糖类物质是影响微囊藻RNA提取的关键因素.为了获得高质量的微囊藻群体总RNA，对比分析4种针对多糖含量较高的方法——方法1 PGTX-bead法、方法2 CTAB-bead法、方法3 FastRNA^® Pro Blue Kit和方法4 RNeasy Mini Kit对微囊藻群体总RNA的提取效果.采用琼脂糖凝胶电泳检测微囊藻群体RNA的完整性，Nanodrop ND1000分光光度计检测RNA纯度及浓度，并采用qPCR检测DNA污染情况.结果表明，4种方法都能从微囊藻群体中提取获得RNA，并在去除DNA后都可以进行RT-PCR等后续实验.方法1 PGTX-bead提取的RNA产量最高，纯度好，DNA污染小，成本低，适合从微囊藻群体中大量提取RNA；方法2 CTAB-bead提取的RNA样品产量也较高，但DNA污染严重，适合需要同时提取DNA和RNA的样本；方法3 FastRNA^® Pro Blue Kit和方法4 RNeasy Mini Kit提取的RNA产量都较低，但方法4操作简单，耗时短，所检测目的基因的相对表达量较高，更适合从少量的微囊藻群体中提取总RNA.;Microcystis colonies are rich in polysaccharide materials which are the key factors influencing RNA extraction. In order to obtain high-quality total RNA from Microcystis colonies, comparative study of several methods including Method 1 PGTX-bead, Method 2 CTAB-bead, Method 3 FastRNA^® Pro Blue Kit and Method 4 RNeasy Mini Kit was conducted. All these methods were suitable for polysaccharide-rich materials. The integrity of RNA was analyzed by agarose gel electrophoresis, RNA concentration and purity were detected by Nanodrop ND1000 spectrophotometer, and DNA contamination was determined by qPCR. The results demonstrated that all the four methods were able to extract RNA from Microcystis colonies, and they were able to be used for downstream experiments, such as RT-PCR, after the contaminated DNA was removed. Method 1(PGTX-bead extraction) yielded RNA of highest concentration, good purity, low DNA contamination and it was low cost; Method 2 (CTAB-bead extraction) also yielded RNA of high concentration, but with heavy DNA contamination. This method was suitable for samples that need simultaneous isolation of RNA and DNA. Method 3 (FastRNA^® Pro Blue Kit)and Method 4 (RNeasy Mini Kit) extraction yielded RNA of low concentration, but Method 4 had simpler protocol, less time consuming, higher relative expression of the detected function gene, thus was suitable for extracting total RNA from small amount of Microcystis colonies.

Abstract 64: Quality and feasibility of a protocol for simultaneous isolation of RNA, DNA and tissue for IHC from needle core lymph node biopsies in DLBCL adapted for multi-center trials.

Abstract Introduction: Personalized medicine strategies targeting specific aberrations unique to an individual tumor, are promising to become a new paradigm in the fight against cancer. Such tailored approaches rely on the thorough profiling of malignant tissues by high-throughput technologies followed by selection of appropriate treatment targeting tumor-specific aberrations. One fundamental challenge, whose resolution is necessary for any successful personalized medicine endeavor, is isolation of useful amounts of good quality molecular material for reliable tumor characterization in downstream assays. Here, we describe our standardized protocol for biopsy collection and results from a multi-center phase II clinical trial investigating the use of panobinostat with or without rituximab in relapsed diffuse large B-cell lymphoma (NCT01238692). Results: Biopsies are performed with patient consent, before treatment initiation and after 15 days of treatment, in patients with safely accessible lesions. Four needle core biopsies are collected at each time point using standard operating procedures to limit pre-analytical variability. Of these, one is preserved in formalin for immunohistochemical (IHC) analyses, while the remaining three are pooled together in RPMI 1640 media. These samples are sent to a central laboratory for further processing. The needle cores in RPMI 1640 undergo B-cell purification using a negative selection kit (StemCell Technologies). Isolation of tumor RNA and DNA is performed using a commercially available kit (Qiagen). Of the 23 patients currently enrolled, 16 (70%) have had a pre-treatment biopsy performed and of these, 7 (44% of those biopsied, 30% of all patients) have also had a post-treatment biopsy, demonstrating the feasibility of tissue collection at participating sites. We assess the effect of transport on the quality and yield of RNA and DNA as well as differences in quality and yield between pre-treatment biopsy vs. biopsies at day 15. The material isolated from these biopsies is evaluated for suitability in downstream applications, namely IHC, gene expression microarray and DNA exome sequencing. Conclusions: The development of standard operating procedures for the collection and processing of biospecimens is essential to control for pre-analytical variability inherent to multicenter trials. Our experience shows that this is both feasible and crucial to understanding of tumor molecular profile changes associated with treatment. Our protocol allows extraction of RNA and DNA of sufficient quality and quantity to permit downstream multi-dimensional analysis. Patient acceptance of research biopsies has been high although the rate of pre-treatment biopsies is greater than that of post-treatment biopsies. Finally, our work demonstrates that the timing of post-treatment biopsies is critical. Citation Format: Torsten H. Nielsen, Zuanel Diaz, Rosa Christodoulopoulos, Lu Yao, Samia Qureshi, Naciba Benlimame, Errol Camlioglu, Michael Crump, Ryan D. Morin, Nathalie Johnson, Tina P. Haliotis, Wilson H. Miller, Sarit Assouline, Koren K. Mann. Quality and feasibility of a protocol for simultaneous isolation of RNA, DNA and tissue for IHC from needle core lymph node biopsies in DLBCL adapted for multi-center trials. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 64. doi:10.1158/1538-7445.AM2013-64

Effect of Smoking Status on Seminal Parameters and Apoptotic Markers in Infertile Men

We assessed semen parameters, sperm apoptotic markers and seminal plasma cotinine in infertile smokers. A total of 160 men were divided into 4 equal groups, including fertile smokers, fertile nonsmokers, infertile smokers and infertile nonsmokers. Smoking was classified as mild--fewer than 10, moderate--10 to 20 or heavy--more than 20 cigarettes daily. All men underwent semen analysis, and assessment of sperm caspase-9, Smac/DIABLO, DNA fragmentation and seminal plasma cotinine. Infertile men, particularly smokers, have significantly lower semen variables and significantly higher sperm Smac/DIABLO, caspase-9 activity, the percent of DNA fragmentation and seminal plasma cotinine. The mean number of cigarettes smoked daily and smoking duration significantly correlated positively with sperm Smac/DIABLO, caspase-9 activity, the percent of DNA fragmentation and seminal plasma cotinine, and significantly correlated negatively with tested semen variables. Heavy smoking was associated with a significant increase in sperm Smac/DIABLO, caspase-9 activity and seminal plasma cotinine, and with a significant decrease in tested semen variables compared with those in moderate or mild smokers. Smoking has a negative impact on semen variables. It is associated with increased sperm caspase-9, Smac/DIABLO and the percent of DNA fragmentation, especially in infertile heavy smokers.

Homemade viral RNA isolation protocol using silica columns: A comparison of four protocols

The Wrst technique for RNA isolation described in the literature was developed by Chirgwin and coworkers in 1979 [1]. They standardized a method for breaking cellular membranes without denaturing nucleic acids through homogenization of tissue in a mixture of guanidinium thiocyanate and -mercaptoethanol followed by an ethanol extraction or ultracentrifugation in a cesium chloride gradient. Although this was a signiWcant advance at that time, the technique consumed a lot of time and was ineYcient, hazardous, and inconsistent. With more recent isolation techniques, the quality, speed, and eYciency of this process have improved. In 1987, Chomczynski and Sacchi improved on the Chirgwin method, converting the guanidinium–hot phenol method to a single-step extraction called the AGPC (acid guanidinium–phenol–chloroform) method [2]. This modiWcation had at least two beneWts: the possibility of processing more samples simultaneously and the isolation of unbroken RNA with a higher purity and yield. Some years later, in 1993, Chomczynski published a reagent for the single-step simultaneous isolation of RNA, DNA, and proteins from cell and tissue samples [3]. Nowadays, there are several nucleic acid isolation kits on the market based on this reagent. More recently, some commercial alternatives have been developed, but it was the development of new techniques employing silica columns for the isolation of nucleic acids

The effect of systemic antibiotics on the microbiological diagnosis of experimental foreign body infections caused by S taphylococcus epidermidis

Qualitative broth culture and quantitative culture on agar were compared with quantitative polymerase chain reaction (PCR) for the diagnosis of foreign body infections (FBI) in a rat model with and without exposure to systemic antibiotics (teicoplanin and rifampin). The 3 methods had a similar and high yield without antibiotics. Antibiotics decreased the number of CFU/foreign body and increased the number of culture-negative foreign bodies and the variability of the results in quantitative culture. The yield of broth culture remained high under antibiotics although prolonged incubation (2-5 days) was required. The yield of the PCR was equivalent or even superior (for teicoplanin) to the yield of broth culture. Quantitative PCR had a higher yield and lower variability than quantitative culture and was not affected by antibiotics. The simultaneous isolation of RNA from all samples indicated viability of the bacteria. Quantitative PCR seems a promising method for the diagnosis of FBI.

Detection of Genomic DNA Fragmentation during Apoptosis (DNA Ladder) and the Simultaneous Isolation of RNA from Low Cell Numbers

In the present paper we describe a rapid and sensitive method for the simultaneous isolation of total RNA and genomic plus low-molecular-weight DNA from apoptotic cells. Using this method, we were able to detect a DNA ladder from as low as 30,000 apoptotic cells in only 45 min including gel electrophoresis. In addition, RNA can be readily obtained from the same specimen to assess gene expression during apoptosis. This method therefore appears to be advantageous when sensitivity and low amounts of sample material are a limiting factor.

The simultaneous isolation of RNA and DNA from tissues and cultured cells.

AbstractMany techniques are currently available that allow the isolation of DNA (1–7) or RNA (8–23), but such methods allow only the purification of one type of nucleic acid at the expense of the other. Frequently, when cellular material is limiting, it is desirable to isolate both RNA and DNA from the same source. Such is the case for biopsy specimens, primary cell lines, or manipulated embryonic stem cells.KeywordsIsoamyl AlcoholGuanidinium ThiocyanatePrimary Cell LineExcess EthanolSterile PolypropyleneThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.