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Automated high multiplex qPCR platform for simultaneous detection and quantification of multiple nucleic acid targets
Louis Hlousek1, Sergey Voronov1, Vesselin Diankov1, Amy B. Leblang1, Patrick J. Wells1, Donna M. Ford*1, Jork Nolling1, Kyle W. Hart1, Patricio A. Espinoza1, Michael R. Bristol1, Gregory J. Tsongalis2, Belinda Yen-Lieberman3, Vladimir I. Slepnev**1, Lilly I. Kong1, and Ming-Chou Lee***1
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Supplementary Material

A plate map, indicating the position and identity of each sample, the assay to be performed, and the results to be reported was constructed using the graphical user interface. The run was initiated following loading of the sample plate, containing PCR mixtures covered with mineral oil, and the CE plate was placed into the instrument. The run, which is fully automated, consisted of an initial denaturation cycle followed by a series of complete cycles of denaturation-annealing-extension, sufficient to amplify any input targets to just below their initial detectability. Next, the metal cannulae of the CE cartridge were immersed directly into the PCRs during the course of one denaturation step. Electrokinetic injection of negatively charged molecules, including DNA amplicons, into the capillaries was performed by applying a fixed voltage during this immersion (11). A very small fraction (estimated < 0.002%) of the sample is transferred per injection. As the TC proceeded to the next annealing step, the cannulae were removed from the reaction wells and transferred to a separate disposable CE plate containing CE running buffer. A fixed voltage of 9kV was then applied for the duration of the electrophoretic separation. Following separation, the instrument automatically purged the separation gel and rinsed the cannulae tips. At this point the TC had gone through two cycles and was entering the denaturation step of a third cycle. The CE cannulae were then reintroduced into the PCRs for the next electrokinetic sampling, which thereafter occurred every other cycle. The run was completed in slightly over 4 h, which included data analyses and reporting.

The capillary array was automatically decontaminated between runs to prevent run-to-run carryover of amplification products.

Measurement of ICEPlex optical detection range

A series of purified random sequence 10-mer oligonucleotides, 5′-labeled with either FAM or TYE-665 (an analog of Cy5; Integrated DNA Technologies, Coralville, IA, USA) at differing concentrations, ranging from 1 × 1013 down to 1 × 107 molecules/L (16.6 M to 16.6 pM) were electrokinetically injected. The areas of the peaks from these oligonucleotides were measured down to background levels.

Measurement of ICEPlex target concentration operating range

The operating range was tested using the ViraQuant assay. Serial dilutions of a single plasmid clone of the BKV target gene (pBK), from 1 × 108 to 10 copies/reaction, were amplified in triplicate in individual wells and quantified using a 48-capillary CE cartridge. The polynomial fit test for linearity was used following Clinical and Laboratory Standards Institute guidelines (12).

Measurement of ICEPlex target size operating range

The range of detectable amplicon sizes was determined by utilizing the Multiplex Ligation-dependent Probe Amplification (MLPA) (13) kit for breast cancer markers (SALSA MLPA kit P078-B1 Breast Tumor; MRC-Holland, Amsterdam, The Netherlands) and genomic DNA from human Jurkat cells (New England BioLabs, Ipswich, MA, USA) following the manufacturer's instructions. The MLPA kit generates 52 amplicons from a variety of genes, spanning the size range of 100 to 500 bp, along with additional control amplicons from 96 bp down to 64 bp. The fragments are separated by 5 to 10 bp with the spacing increasing for the larger molecules. The reaction end points were placed into the instrument and electrokinetically injected to establish the separation capabilities of the system.

Demonstration of ICEPlex two-color capability

A series of primer pairs, with target Tm values of approximately 60°C, was developed to create a 16-target, two-color multiplex assay derived from the AlloMap gene expression test from XDx (Brisbane, CA, USA). Nine genes were targeted using FAM-labeled primers, and seven genes were targeted using TYE-labeled primers.

Donor whole blood was collected into BD vacutainer CPT heparin tubes (Becton Dickinson, Franklin Lakes, NJ, USA) to separate white blood cells. These were then lysed, and the RNA extracted using QIAshredder and RNeasy extraction kits from Qiagen following the manufacturer's instructions. cDNAs were generated with random hexamer priming and Superscript reverse transcriptase (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. Final products were amplified with the above described primers along with 2× Qiagen Multiplex PCR Master Mix with HotStarTaq DNA polymerase and analyzed on the ICEPlex system.

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