Quantitative assay for submicrogram amounts of protein

An ultrasensitive protein assay, linear between 0.01 and 0.2 μg protein, is described. In this assay, copper is complexed to protein, excess copper is removed by adsorption to a small Sephadex column, and the copper-protein complex is destroyed by digestion with hydroperoxide. Phenol and chloramine-T are added to the reaction mixture and the copper catalyzes the production of a color-producing reaction between the two compounds. The assay is not affected by low levels of phosphate, Tris, metals, or a tenfold excess of nucleic acid over protein. Reducing agents, sucrose, certain anions, high salt concentrations, and EDTA seriously interfere. The method is about 500 times as sensitive as that of Lowry et al. [Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951)J. Biol. Chem.193, 265]. Like the biuret procedure, the assay measures copper complexed to the peptide bonds but is probably influenced by other factors, since equivalent amounts of different proteins give similar but not identical amounts of color.     

Strategies for microarray analysis of limiting amounts of RNA.

One of the critical limitations of current microarray technologies for use in expression analyses is the relatively large amount of input RNA required to generate labelled cDNA populations for array analysis. In situations where RNA is limiting, the options for expression profiling are to increase cDNA labelling and hybridisation efficiency, or to use an amplification strategy to generate enough RNA/cDNA for use with a standard labelling method. Sample amplification approaches must preserve the representation of the relative abundances of the different RNAs within the starting population and must also be highly reproducible. This review evaluates current signal and sample amplification technologies, including those that can be used to generate labelled cDNA populations for array analysis from as little as a single cell.     

改良异硫氰酸胍一步法提取红豆杉细胞RNA

将Chomczynski建立的RNA一步分离法加以适当改进,用以提取红豆杉细胞株E2、TC、H21的总RNA,所获RNA样品的A260/A280比值分别是1.82±0.03、1.85±0.04、1.74±0.03;A260/A230比值分别为2.02±0.03、2.15±0.03、2.03±0.05;得率分别为51.36±0.05μg/g、52.40±0.04μg/g、51.08±0.06μg/g新鲜细胞.变性琼指糖凝胶电泳图谱显示每一样品均具有完好的28S和18S两条亮带.     

Simple and effective method for generating single-stranded DNA targets and probes

A simple and efficient PCR method was developed for generating dye- or radiolabeled single-stranded DNA targets or probes used for hybridization studies. The method involved the use of a pair of long primers with high annealing temperatures and a short, labeled primer with a low annealing temperature in a PCR consisting of two cycles at different temperatures. We used this method to generate dye Cy 5-labeled and [32P]-radiolabeled single-stranded DNA targets and probes. These labeled probes were used successfully for the microarray identification of point mutations in Mycobacterium tuberculosis genes and for the Northern blot detection of expression changes of the GATA-2 gene in Pneumocystis carinii-infected rat lungs.     

Long-lived southern blots using RNA probes

Conditions for preparation and hybridization of Southern blots are described which assure reusability through 15 to 25 cycles. The procedure relies on the use of charge-modified nylon membranes and^[32P]-labelled RNA probes.     

总RNA和mRNA来源的探针与cDNA芯片杂交的差异研究

提取BEP2D细胞的总RNA并按两种方式进行cDNA芯片探针的标记,一种是将100μg BEP2D细胞的总RNA利用逆转录法直接标记成荧光探针,另一种是先从100μg BEP2D细胞的总RNA中分离出mRNA,然后再标记成荧光探针。将两份标记好的探针同时与含有230个基因的cDNA芯片杂交。杂交后的芯片经Axon4100B扫描仪扫描,发现两种方式标记探针的一致性为93．04％,并且mRNA来源探针杂交后的荧光信号值较总RNA的弱。探讨了这两种方法标记探针在基因芯片表达谱研究中的差异性,目的是为利用这两种方法标记探针进行基因表达谱研究提供一些依据。     

CTAB-urea method purifies RNA from melanin for cDNA microarray analysis

Melanin represents a major problem for the study of melanoma by microarrays since it is retained during RNA extraction and inhibits the enzymatic reactions used for probe preparation. Here we report a new method for cleaning RNA from melanin, based on the use of the cationic detergent cetyl-trimethylammonium bromide (CTAB)-urea for RNA precipitation. This method is easy to perform and has a low cost. Purified RNA is recovered with high quality and good yield. CTAB-urea treated RNA from highly pigmented melanoma cells can be successfully reverse transcribed and labeled to obtain probes which can be subsequently used in cDNA microarray experiments, giving consistent and reproducible results.     

Quantitation of submicrogram amounts of protein using coomassie brilliant blue R on sodium dodecyl sulfate-polyacrylamide slab-gels.

A sodium dodecyl sulfate (SDS)-polyacrylamide slab-gel system was used to study the use of Coomassie brilliant blue (CB) as a quantitative stain. Quantitation curves are shown for tubulin, cytochrome c, histone, and actin from gels stained with CB. A comparison was made of three identical gels using an actin sample and stained with CB, fast green and buffalo black. CB staining was found to be quantitative in the 0.05–2.75-μg range as well as possessing an order of magnitude increase in sensitivity over other stains tested.     

A method for generating selective DNA probes for the analysis of C-negative regions in human chromosomes

Linker-adapter polymerase chain reaction (LA-PCR) is among the most efficient techniques for whole genome DNA amplification. The key stage in LA-PCR is the hydrolysis of a DNA sample with restriction endonucleases, and the choice of a restriction endonuclease (or several endonucleases) determines the composition of DNA probes generated in LA-PCR. Computer analysis of the localization of the restriction sites in human genome has allowed us to propose an efficient technique for generating DNA probes by LA-PCR using the restriction endonucleases HaeIII and RsaI. In silico hydrolysis of human genomic DNA with endonucleases HaeIII and RsaI demonstrate that 100- to 1,000-bp DNA fragments are more abundant in the gene-rich regions. Applying in situ hybridization to metaphase chromosomes, we demonstrated that the produced DNA probes predominantly hybridized to the C-negative chromosomal regions, whereas the FISH signal was almost absent in the C-positive regions. The described protocol for generating DNA probes may be successfully used in subsequent cytogenetic analysis of the C-negative chromosomal regions.     

Increased yield of total RNA from fine-needle aspirates for use in expression microarray analysis

Fine-needle aspirate samples hold the potential for gaining valuable insight into the molecular details and prognostic indicators for certain types of cancer in a limited volume of relatively pure tumor cells. Although limited, such clinical samples can be used with high efficiency when analyzed in conjunction with gene-dense expression microarrays. For this reason, it is essential to retrieve as much high-quality genetic material as possible from each fine-needle aspirate sample. We have conducted a study to improve the efficiency of extracting high quality total RNA to use in microarray analysis from single ex vivo fine-needle aspirate samples of 11 breast cancers added to RNAlater RNA Stabilization Reagent immediately upon collection. Approximately half the total RNA from fine-needle aspirate samples of breast cancers was isolated from the supernatant, and that RNA had similar quality and gene expression profile to the RNA that was isolated from the corresponding cell pellet. We recommend that the supernatant not be discarded when extracting RNA from fine-needle aspirate samples stored in RNAlater.