The real-time quantitative polymerase chain reaction (rtqPCR) has overcome the limitations of conventional, time-consuming quantitative PCR strategies and is maturing into a routine tool to quantify gene expression levels, following reverse transcription (RT) of mRNA into complementary DNA (cDNA). expression of individual cells and thus could complement global microarray-based expression profiling strategies. INTRODUCTION Gene expression is regulated at the level of individual cells, with different cell types or different developmental stages of the same cell expressing distinct sets of genes. Thus, analysis of the gene expression pattern of individual cells is a desirable goal. Laser-based microdissection techniques enable the isolation of identified single cells from fixed tissue (1,2), while the patch-clamp method enables the harvesting of mRNAs from a single living cell (3,4). The latter technique PKI-587 manufacturer is of particular importance as it permits correlation PKI-587 manufacturer of the functional properties of individual cells with their specific gene expression profile (5). Reverse transcription of mRNA followed by polymerase chain reaction (RTCPCR) is a reliable method of detecting gene expression. To analyze mRNA expression from single cells without loss of low abundance mRNAs, it is common practice to use all of the total single-cell complementary DNA (cDNA) reaction mixture as template PKI-587 manufacturer for subsequent PCR (3). It is of increasing importance to detect not only qualitative but also quantitative differences in gene expression levels, and several approaches have been used to obtain quantitative data from single-cell RTCPCR experiments. These include the addition of known amounts of competitor mRNA (6,7), serial dilution of single-cell cDNA pools (8), and fluorescence-based real-time quantitative PCR (rtqPCR) (9). The latter has the advantage that it is easy to perform, highly reproducible, and, importantly, has the sensitivity to amplify and quantify even a single DNA template molecule (10C14). Thus, rtqPCR has become the method of choice for quantitative analysis of gene expression levels (14C17). However, I and others have observed that components of the undiluted RT reaction mixture considerably distort the subsequent PCR amplification reaction, presumably by inhibiting at 4C) for 15 min, the supernatant was removed. The cDNA pellet was dried in a thermal heating block (Eppendorf) Rabbit Polyclonal to NDUFA3 at 45C until all ethanol had evaporated, and then PKI-587 manufacturer dissolved in 10 l of sterile water (Eppendorf). In order to completely resolubilize the cDNA, it was incubated for 60 min at 45C prior to quantitative real-time PCR. TaqMan quantitative real-time PCR and data analysis rtqPCR was performed as recently described (13) using the GeneAmp 5700 instrument (Applied Biosystems). The TaqMan primer/hybridization probe real-time PCR approach uses a fluorescence resonance energy transfer probe as reporter system (24). Hybridization primer/probe assay specific for real-time PCR detection of TH (accession no. “type”:”entrez-nucleotide”,”attrs”:”text”:”M69200″,”term_id”:”201997″,”term_text”:”M69200″M69200) was optimized according to the recommended criteria using the Abiprism Primer express software (Applied Biosystems) and the 2 2 TaqMan hybridization-probe Mastermix (Applied Biosystems). The TaqMan Mastermix contains uracil-= 10(C1/slope) for serial dilution in steps of 10 [log(10) scale] or = 2(C1/slope) for serial dilution in steps of 2 [log(2) scale]. = 2 reflects a doubling of DNA in each PCR cycle over all dilution steps. Errors are given as standard deviations (SDs) of the means. Significance was defined according to = 5 experiments: C3.35 0.09, = 1.99 0.04). This slope indicates a near perfect doubling of amplification products per cycle during the exponential phase of the PCR [the theoretical slope for an ideal PCR amplification is C1 / log(10)2 = C3.32]. Open in a separate window Figure 1 Real-time fluorescent RTCPCR standard curve for TH cDNA quantification. Top, sensitivity of the real-time fluorescent RTCPCR protocol for TH. Relative fluorescence intensities (= 0.999) was highly reproducible (mean slope = C3.31 0.09 SD, = 3) and defined the intercept at PCR cycle = 40.76 for a single TH dsDNA molecule. When undiluted single-cell cDNA was used as PCR template, the results were dramatically different from those obtained using purified DNA (Fig. ?(Fig.1),1), or cDNA in water (see Fig. ?Fig.4A).4A). The rtqPCR amplification kinetics varied from cell to cell in an unpredictable fashion, making the definition of the cycle threshold for detection problematic (Fig. ?(Fig.2A).2A). These results appear to rule out reliable rtqPCR quantification of cDNA molecules using undiluted single-cell cDNA reaction mixtures as PCR templates. Open in a separate window Figure 2 Real-time PCR using undiluted single-cell cDNA as template. (A) Amplification plots for TH rtqPCR for three individual cells utilizing undiluted cDNA reactions as templates. Relative fluorescence intensities (= 6) for cDNA in water; = 4) for cDNA in patch-clamp buffer; = 4) for cDNA in 2 U/l RNasin; = 4) for cDNA in RT buffer; = 4); = 6) for cDNA in reverse transcriptase. A two-paired 0.000005) and to a.