and each specimen was collected during the pH1N1 epidemic in Boston. One limitation of the bench top RT-PCR used in this study was that the assays were performed on the clinical specimens after they had undergone at least one freeze/thaw cycle, which may have led to some RNA degradation in the sample and thus slightly reduced the sensitivity of the gold standard RT-PCR. We hypothesize that this could potentially explain the two DFA-positive, bench top PCR-negative results. The DFA negative/bench top RT-PCR positive specimen was one which was already known to have low numbers of epithelial cells and was accordingly borderline inadequate for DFA testing; a separate specimen from the same patient had tested positive for pH1N1 by RT-PCR testing done at the Massachusetts State Laboratory, confirming our own bench top RT-PCR results. We plotted the input viral load as determined by the bench top quantitative PCR against the amplicon output concentration from the microfluidic assay measured using capillary electrophoresis. Since these were endpoint reads, we did not RO4929097 expect a strong linear relationship between input copy number and endpoint concentration, and we did not see one. All of the microfluidic assays were run with the same primer, enzyme and blocking agent starting concentrations. These concentrations were chosen based on a series of preliminary optimization experiments that are described in the data. The only variable in the reaction was the input sample, which varied in several ways. First, there was variation in the viral load between specimens. There were also differences in sample type and in the method of storage before microfluidic testing. The samples collected at BMC were aliquotted and stored in VTM, while almost all of the samples collected at BIDMC were not aliquotted and were stored in PBS. The aliquotting allowed for minimal freeze thaws before testing the samples in the reference bench top RT-PCR assay and the microfluidic chip assay. Most of the samples went through one freeze/thaw cycle before microfluidic testing and the bench top RT-PCR. In addition, all samples were spun once before introduction into the microfluidic chip. The BMC samples were spun before aliquots were frozen, and the BIDMC samples were spun after aliquots were thawed fore testing in the microfluidic assay. This ��preprocessing��step was performed to minimize the chance of clogging the nucleic acid extraction part of the chip with cell debris, which was seen in a small number of the preliminary experiments due to the very small pore size of the SPE columns used in these chips. Recent reformulations of the SPE in 
our lab have resulted in larger average pore sizes that have eliminated the need for this initial spin down step. It is possible, however that the spin step also acted to PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22189542 remove PCR inhibitors, resulting in better results than had the samples been left completely untreated. The PCR channel design used in the microfluidic assay was optimized previously to achieve repeatable and predictable temperature profiles in the chip. The serpentine PCR channel flows the sample through 30 cycles of PCR. The cycle number is fixed for any given chip, unlike in bench top PCR schemes. Increasing the cycle number and/or reducing the flow rate could further improve the PCR yield, but testing time would also increase. So, for any given assay, the chip design and the PCR reaction itself must be optimized simultaneously. Here, a validated protocol obtained from th
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