Methods 

Abstract: The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data.

Abstract: Normalization means to adjust microarray data for effects which arise from variation in the technology rather than from biological differences between the RNA samples or between the printed probes. This paper describes normalization methods based on the fact that dye balance typically varies with spot intensity and with spatial position on the array. Print-tip loess normalization provides a well-tested general purpose normalization method which has given good results on a wide range of arrays. The method may be refined by using quality weights for individual spots. The method is best combined with diagnostic plots of the data which display the spatial and intensity trends. When diagnostic plots show that biases still remain in the data after normalization, further normalization steps such as plate-order normalization or scale-normalization between the arrays may be undertaken. Composite normalization may be used when control spots are available which are known to be not differentially expressed. Variations on loess normalization include global loess normalization and two-dimensional normalization. Detailed commands are given to implement the normalization techniques using freely available software.

Abstract: Those who have worked in the field of quantitative polymerase chain reaction (PCR) since the early 1990s have accepted many of the tedious aspects of the early assays as routine. Difficulties associated with early quantitative PCR techniques included: (i) ensuring that the PCR was within the linear range of amplification (that portion where the PCR signal is directly proportional to the input copy number) and (ii) finding a suitable method to detect the product once linear amplification was achieved. To ensure linearity, researchers would amplify serial dilutions of cDNA (1) or alter the amplification cycle for each gene (2). Others would add a competitor template (3), perform limiting dilution assays (4), or use a PCR mimic with a similar primer sequence that would be coamplified along with the product (5). Detection of the amplicon generally resorted to running gels and DNA detection using a stain, radioactivity, or probe. For those who have converted to using real-time quantitative PCR, the old days are but a memory. In real-time PCR, worrying about linear amplification, running gels, working with radioactivity, and gathering numbers are a thing of the past. The ability to generate data within a 2to 3-h period is now a reality. Other advantages of real-time PCR include enhanced sensitivity, high throughput, use of a closed-tube system, reduced variation, the ability to simultaneously multiplex reactions, and lack of post-PCR manipulations. The technology to detect PCR products in real time, i.e., during the reaction, has been available for more than 5 years, but has seen a dramatic increase in use over the past 2 years. A Medline search using the key words TaqMan or real-time and PCR yielded 19 citations in 1996, 28 citations in 1997, and 52, 157, and 409 citations in 1998, 1999, and 2000, respectively. At the time of this writing, there were 551 citations in 2001 (Fig. 1). Various manufacturers are touting realtime PCR instrumentation and affiliated technology

Abstract: Identification of components present in biological complexes requires their purification to near homogeneity. Methods of purification vary from protein to protein, making it impossible to design a general purification strategy valid for all cases. We have developed the tandem affinity purification (TAP) method as a tool that allows rapid purification under native conditions of complexes, even when expressed at their natural level. Prior knowledge of complex composition or function is not required. The TAP method requires fusion of the TAP tag, either N- or C-terminally, to the target protein of interest. Starting from a relatively small number of cells, active macromolecular complexes can be isolated and used for multiple applications. Variations of the method to specifically purify complexes containing two given components or to subtract undesired complexes can easily be implemented. The TAP method was initially developed in yeast but can be successfully adapted to various organisms. Its simplicity, high yield, and wide applicability make the TAP method a very useful procedure for protein purification and proteome exploration.

Abstract: The three-dimensional culture of MCF-10A mammary epithelial cells on a reconstituted basement membrane results in formation of polarized, growth-arrested acini-like spheroids that recapitulate several aspects of glandular architecture in vivo. Oncogenes introduced into MCF-10A cells disrupt this morphogenetic process, and elicit distinct morphological phenotypes. Recent studies analyzing the mechanistic basis for phenotypic heterogeneity observed among different oncogenes (e.g., ErbB2, cyclin D1) have illustrated the utility of this three-dimensional culture system in modeling the biological activities of cancer genes, particularly with regard to their ability to disrupt epithelial architecture during the early aspects of carcinoma formation. Here we provide a collection of protocols to culture MCF-10A cells, to establish stable pools expressing a gene of interest via retroviral infection, as well as to grow and analyze MCF-10A cells in three-dimensional basement membrane culture.