Extraction of total RNA from adipocytes.

RNA isolation from adipocytes presents with several technical problems and yields unacceptable results when following standard protocols. Here, we will describe additional steps and modifications necessary for the use of different RNA isolation protocols in terms of RNA yield, RNA quality and preparation time. Using five times the recommended quantity of lysis buffer, incubating the lysate at 37 degrees C, repeatedly passing the lysate through a cannula, and centrifugation to remove the lipid layer are essential additional steps when working with adipocytes. With these modifications, isolation of total RNA resulted in an average yield of 12-30 microg total RNA from 2 x 10(6) cells. Preparation times were similar for all but the CsCl gradient method. The purest RNA was obtained by spin-column purification, whereas acid phenol-chloroform methods yielded the highest amounts of total RNA. CsCl gradient ultracentrifugation is suggested for situations where DNase I digestion is impractical.     

An improved method for the quantitative isolation of rat liver nuclear RNA polymerases.

Rat liver nuclear RNA polymerases exist in two functional states, one of which is active towards the endogenous chromatin template (engaged enzyme), while the other is inactive (free enzyme) (Yu, F.L. (1974) Nature 251, 344-346). This paper reports the direct separation of these two populations of RNA polymerases from isolated rat liver nuclei by a simple extraction procedure. It is estimated that as much as 50% of the total nuclear RNA polymerase activity in normal rat liver may exist in the form of the free enzyme. Evidence is also presented to indicate that the free enzyme activity is easily lost when the nuclear isolation procedure involves the use of an isotonic buffer medium, or when the isolated nuclei are subjected to sonication as is required for the solubilization of the nuclear RNA polymerases by the conventional method. Based on these new findings, it is proposed that nuclei be isolated directly in hypertonic sucrose and that the free enzyme be extracted before the nuclei are subjected to sonication to solubilize the engaged enzyme. This method circumvents the loss of the free RNA polymerase population and, as a result, the total yield of the nuclear RNA polymerases is greatly increased. The possible functional role of the free RNA polymerase in gene expression is discussed.     

Impact of preanalytical handling and timing for peripheral blood mononuclear cells isolation and RNA studies: the experience of the Interinstitutional Multidisciplinary BioBank (BioBIM)

Multicenter studies and biobanking projects require blood transportation from the participating center to a central collection or diagnostic laboratory. The impact of time delays between venous blood collection and peripheral blood mononuclear cells (PBMC) isolation prior to RNA extraction may affect the quality and quantity of isolated nucleic acids for genomic applications. Thus, standard operating procedure (SOP) optimization for the treatment of biological samples before RNA extraction is crucial in a biological repository. In order to define SOPs for whole blood preservation prior to RNA extraction, we sought to determine whether different blood storage times (0, 3, 6, 10, 24, and 30 hours) prior to PBMCs isolation and storage at -80°C, could affect the quality and quantity of extracted RNA. After spectrophotometric quantification, the quality and integrity of RNA were assessed by agarose gel electrophoresis, RNA integrity number and real time-PCR (RT-PCR).?Across the different time points we did not observe significant differences within the first 24 hours of blood storage at room temperature, while a significant loss in RNA yield and integrity was detected between 24 and 30 hours. We conclude that time delays before PBMCs isolation prior to RNA extraction may have a significant impact on downstream molecular biological applications.     

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.     

Isolation of Functional RNA from Periderm Tissue of Potato Tubers and Sweet Potato Storage Roots

A reliable and efficient protocol is given for the isolation of mRNA from the periderm of potato tubers and sweet potato storage roots. The method relies on a urea-based lysis buffer and lithium chloride to concentrate total RNA away from most of the cytoplasmic components and to prevent oxidation of phenolic complexes. To enhance the physical separation of the RNA from other macromolecular components, the RNA fraction was incubated in the presence of the cationic surfactant Catrimox-14. Poly(A)⁺ mRNA was separated from total RNA and other contaminants by using Promega's MagneSphere technology. The mRNA was suitable for cDNA library construction and RNA fingerprinting.     

Improved RNA isolation from cells in tissue culture using a commercial nucleic acid extractor.

The Applied Biosystems 340A Nucleic Acid Extractor automates isolation of either DNA or RNA from tissue or cells in culture. We have found that several modifications to the manufacturer's recommended protocol greatly improve the quality of RNA that can be routinely isolated from cells in culture. These modifications include lysis of monolayer cells directly on plates, centrifuging samples after homogenization to remove precipitable RNase contaminants and purging the instrument's reagent lines with 0.1% diethyl pyrocarbonate. These simple modifications enhance both RNA quality and reproducibility of yield.     

Separation of live cells in different phases of the cell cycle for gene expression analysis

BACKGROUND: Homogeneity of cell populations is a basic requirement for gene expression analyses of the cell cycle, such as those based on microarrays. The most common approach to obtain specific populations is the use of synchronization methods that increase the number of cells representing a certain cell cycle stage. On the one hand, conventional synchronization usually causes undesirable effects. On the other hand, cell separation methods may imply loss of RNA quality, another limiting factor for expression profiling. We describe a new strategy to specifically separate live cells in different phases of the cell cycle (G(1) and G(2)/M) to obtain good quality RNA for gene expression analyses. METHODS: The experimental design included sorting G(1) and G(2)/M cells with the vital fluorochrome Hoechst 33342, followed by RNA isolation from the sorted cells. RESULTS: Sorted living G(1) and G(2)/M cells, analyzed by immunocytochemistry and laser scanning cytometry, showed strong enrichment. The quality and specificity of the isolated RNA were demonstrated by northern blot. CONCLUSIONS: This new approach has many potential applications, such as expression profiling of specific cell populations after eliminating the irrelevant data produced by cells in other stages of the cycle.     

RNA preservation of Antarctic marine invertebrates

Fifteen species of marine invertebrate commonly occurring in the near-shore environment of Rothera base, Antarctica, were used to test tissue sample storage protocols with regard to preservation of RNA integrity. After animal collection, the tissues were either immediately extracted for RNA or stored at −80°C after having been, either directly flash frozen in liquid nitrogen or preserved in a commercial RNA storage solution, for extraction in the UK. In four cases, direct flash freezing produced enhanced RNA integrity compared with samples in the commercial storage solution. A subset of samples were further tested for the preferred temperature of storage in the commercial reagent. RNA integrity was well preserved at both +4 and −20°C over periods of 2 months, but degradation was rapid in tissues stored at room temperature. Eight out of the fifteen species only produced a single ribosomal band on gel electrophoresis. This survey provides a guide for tissue transport of Polar cold water marine invertebrates.     

Isolation of high-molecular-weight DNA from small samples of blood having nucleated erythrocytes,collected, transported,and stored at room temperature

Blood samples collected in the field for isolating DNA suitable for molecular analysis need special care in their storage and handling. In this article, we describe a simple method for the isolation of good-quality high-molecular-weight DNA that does not require low temperature conditions during collection, storage, and/or transportation of blood samples. This method involves smearing small aliquots of blood onto clean slides and air drying them at room temperature. The slides with blood smears can then be transported or stored at room temperature and still serve as a very good source of high-molecular-weight DNA. Genomic DNA from these samples can be extracted by organic phase separation (phenol-chloroform extraction) after lysis. The DNA thus obtained is of high quality and yields DNA fingerprints qualitatively similar to those prepared from corresponding control DNA isolated from frozen blood samples. Needing minimal facilities at field sites, the method is very convenient for conducting RFLP analysis of wild/field populations for demographic, behavioral, and ecologic studies.     

Enrichment of C-type virus mRNA from immunochemically separated polyribosomes.

A method for the isolation of mRNA from a c-type virus-positive cell (JLS-V6) has been developed. This method consisted of (i) specific immunochemical precipitation of polyribosomes synthesizing viral proteins, (ii) extraction of the mRNA from the polyribosome/antigen/antibody complex, and (iii) separation of poly-A containing RNA by affinity chromatography on oligo-d(T)-cellulose. Three size classes of virus specific poly-A containing mRNA are identified and they are 35S, 20S-25S, and 10S-15S.     

红酵母RY-4中虾青素的分离纯化研究

本文主要研究了红酵母RY-4菌株中天然虾青素的提取和纯化工艺。通过各种破壁提取方法的比较分析,得出了酸热法破壁丙酮提取为较经济理想的粗色素提取方法,提取的粗色素油中虾青素含量达51.7%。通过对各种柱层析条件的摸索研究,得出了以硅胶为吸附剂,Ⅴ(石油醚)∶Ⅴ(乙酸乙酯)混和液为洗脱剂梯度洗脱从粗色素油中分离纯化虾青素的方法,所得虾青素纯度达到97%。