Tom Morrison

Continuous fluorescence observation of amplifying DNA allows rapid and accurate quantification of initial transcript copy number. A simple and generic method for monitoring product synthesis with the double-stranded DNA dye, SYBR Green I provides initial template copy number estimation limited only by stochastic effects. To reach this degree of sensitivity, two methods were used. First, specific products generally have a higher melting temperature than nonspecific products, and therefore, specific product formation was monitored by fluorescence acquisition at temperatures at which only specific products are double-stranded. Second, anti-Taq antibodies were used to reduce nonspecific product generation. The log-linear portion of the fluorescence vs. cycle plot was extended to determine a fractional cycle number at which a threshold fluorescence was obtained. These fractional cycle numbers were plotted against the log of starting template copies to give linear standard curves from purified PCR products, allowing easy estimation of cDNA unknowns over a 10(6)-fold range. A single template molecule per reaction could be distinguished from the absence of template, although stochastic effects increased the variance of concentration estimates below 10 copies. Above 10 copies per reaction, typical replicate coefficients of variation were 6%-37%, with better precision at higher copy numbers.

Understanding biological complexity arising from patterns of gene expression requires accurate and precise measurement of RNA levels across large numbers of genes simultaneously. Real time PCR (RT-PCR) in a microtiter plate is the preferred method for quantitative transcriptional analysis but scaling RT-PCR to higher throughputs in this fluidic format is intrinsically limited by cost and logistic considerations. Hybridization microarrays measure the transcription of many thousands of genes simultaneously yet are limited by low sensitivity, dynamic range, accuracy and sample throughput. The hybrid approach described here combines the superior accuracy, precision and dynamic range of RT-PCR with the parallelism of a microarray in an array of 3072 real time, 33 nl polymerase chain reactions (RT-PCRs) the size of a microscope slide. RT-PCR is demonstrated with an accuracy and precision equivalent to the same assay in a 384-well microplate but in a 64-fold smaller reaction volume, a 24-fold higher analytical throughput and a workflow compatible with standard microplate protocols.

The quantity of Borrelia burgdorferi organisms in tissue samples is an important determinant for infection studies in the mouse model of Lyme disease. This report presents the development of a rapid and sensitive external-standard-based PCR assay for the absolute quantification of B. burgdorferi in mouse tissue samples. The assay uses a double-stranded DNA dye to continuously monitor product formation and in less than an hour was able to quantify samples ranging up to 6 log units in concentration. The PCR efficiencies of the sample and the standard were matched by using a standard composed of purified B. burgdorferi chromosome mixed with tissue-matched mouse genome lacking bacterial DNA. Normalization of B. burgdorferi quantities to the mouse nidogen gene allowed comparison of B. burgdorferi numbers in samples isolated from different tissues and strains. PCR analysis of the chromosomal gene recA in cultured B. burgdorferi was consistent with a single recA per bacterium. The parameters defined in this assay should be applicable to quantification of other organisms, even infectious agents for which no ready source of DNA standard is available. In summary, this report presents a rapid external-standard-based PCR method for the quantification of B. burgdorferi in mouse DNA samples.

Borrelia burgdorferi possesses membrane lipoproteins that exhibit stimulatory properties and, consequently, have been implicated in the pathology related to Lyme disease. As CD14 has been shown to mediate signaling by a number of lipid-modified bacterial products, the involvement of CD14 in signaling mediated by two B. burgdorferi lipoproteins, outer surface protein A (OspA) and OspC, was determined. Lipoprotein-mediated induction of nuclear factor-kappaB nuclear translocation and production of IL-8 and IL-6 in HUVEC was enhanced in the presence of serum or soluble rCD14. CD14-specific Abs that block LPS-mediated signaling also inhibited lipoprotein-dependent signaling in HUVEC and neutrophils. The formation of stable complexes between OspA and CD14 was demonstrated by native gel electrophoresis. LPS was found to compete with OspA for binding with CD14, suggesting that LPS and OspA bind similar regions on CD14. The similarity in binding was further supported by the finding that a mutant soluble CD14, lacking the LPS binding site, did not facilitate lipoprotein signaling, nor did it form a complex with OspA. Binding of OspA to CD14 was dependent on the lipid modification, as unlipidated OspA did not form a complex with CD14 or stimulate cells. In contrast, the lipopeptide remaining after proteinase K digestion both formed a complex with CD14 and retained stimulatory properties. These findings indicate that CD14 facilitates bacterial lipoprotein signaling in mammalian cells.