Pushing the limits of real time PCR
Applied Biosystems and ITN’s model partnership get results

apid and reproducible gene expression studies are a critical piece of the ITN’s search for biomarkers of immune tolerance. A combination of wide dispersal, genome-wide techniques and more targeted, quantitative expression analysis would provide a highly efficient means of identifying potential gene candidates and assessing their value. Considering that the ITN currently has 20 trials contributing clinical specimens to the effort, high-throughput facilities are a definite “must-have”.

Enter Applied Biosystems (ABI) of Foster City, California. ABI, a business group of Applera Corporation, is an ITN technology partner providing high-throughput, real time PCR services to the ITN Gene Expression and Profiling Core Facility.

Overseeing the technical aspects of the ITN collaboration is Kathryn Hunkapiller, Gene Expression and Genotyping Services Production Manager at ABI. Ms. Hunkapiller began as in intern at ABI in the summer of 1995. After receiving her Master of Science degree in Biology from Stanford, she became a full-time ABI employee.

Applied Biosystems pioneered the use of fluorescent nucleotide labeling, leading to the advent of automated sequencers in the mid-1980s. Later, in the mid-1990s, the company went on to develop the first real-time PCR analysis instrument.

Hunkapiller’s team is adept at optimizing any PCR – especially critical when it comes to probing scarce human tolerance genes. Because the ITN is running so many mechanistic studies for each trial, ensuring adequate specimen for each study is a continual struggle. This project is also especially challenging since some of the so-called “dark side genes” of human tolerance – those suspected tolerance genes that are present, but for which there is no evidence of expression – are included in the ITN library.

To address this ongoing issue, Hunkapiller and her team have been working with a new concept, pre-amplification, to reduce the initial amount of RNA required for PCR. Hunkapiller’s favored pre-amp protocol consists of 10 PCR cycles using all primer sets of interest. So far, she’s been able to squeeze all ITN primer sets into a single reaction – that’s up to 1000 sets of primers and probes per tube.

“We call it kiloplexing,” says Hunkapiller. “Before pre-amplification, we averaged about 50% detection with ITN samples. With pre-amplification, we’ve been able to increase that to about 85%. Also, we can start with 700ng rather than 30 µg.” Hunkapiller has converted all ITN reactions to the pre-amp method in order to increase the sensitivity of the reaction.

“Low expressors,” as she calls them, are a key focus of Hunkapiller’s work with the ITN. Establishing a reliable and consistent baseline and detecting even small deviations in expression are the goals of the core’s thorough QA/QC program.

QA starts with the ITN Specimen Repository Core – McKesson BioServices in Rockland, Maryland – where whole blood specimens are processed into RNA samples. These high-yield protocols, developed in collaboration with the ITN Tolerance Assay Group and McKesson scientists, are conducted with highly standardized procedures, producing samples of very high purity RNA.

Hunkapiller’s group conducts their own QA/QC battery upon receipt of ITN samples – these tests include UV concentration and purity analysis as well as 18s and 28s band integrity. Each sample is then amplified and analyzed in quadruplicate. Reagents and equipment are checked routinely – everything from sample viscosity to robot calibration is optimized on a pre-determined schedule. Currently, the Applied Biosystems Integrated Lab Services Business, to which the ITN lab has contributed its expertise, is performing additional R&D to further increase efficiency of the QA processes.

Applied Biosystem's ABI PRISM® 7900HT Sequence Detection System

Hunkapiller’s state-of-the-art lab currently operates six second-generation ABI PRISM® 7900HT Sequence Detection Systems, each capable of processing ten 384-well microtiter plates per day – that’s close to 1000 samples per day. The lab also features several robots, a MATRIX 2D barcode system and three restricted-access amplicon-free rooms. Since a technician can process about 16 RNA samples per day, all 16 samples can go through QC and be screened in quadruplicate for all 1000 ITN target genes within a week. Hunkapiller’s lab recently analyzed over 300 samples from Dr. Peter Creticos’ ITN study of AIC in sufferers of ragweed allergy.

The ITN collaboration with ABI is viewed by many in the ITN as a model partnership. The advantages to the ITN are evident in the quality of the data generated by the core and the high volume of samples that can be processed under the partnership. For Applied Biosystems, the collaboration has added to their product line, with primer-probe sets developed for the ITN now available worldwide through their TaqMan® Assays-on-Demand™ Gene Expression products – pre-formulated, ready-to-use probe and primer sets. More importantly, perhaps, the ITN studies have offered an unparalleled opportunity for real-world testing of their products, resulting in improved protocols and a wealth of new performance data.

“With de novo sequencing, give people and A, T, G or C and they’re happy,” says Hunkapiller. “Functional genomics is not so cut-and-dry. More ambiguity exists in determining the function of a gene and, therefore, a need for a more intimate relationship with the customers is required to understand the specific biological problems that each customer is trying to solve. The ITN collaboration is allowing us to get closer to the science.”