Renewing the Promise of Real-time PCR

Dr. Brian Caplin realized that the promise of real-time PCR as an easy genotyping and quantification technology had been broken and a gap existed in real-time PCR technology. End-users of real-time PCR were being hindered by the time and effort necessary to design and optimize their experiments.
Dr. Caplin launched Fluoresentric to fill that gap by providing expert design and optimization of real-time PCR, allowing researchers and technicians more time for collecting the data necessary to advance their work.

What makes Real-Time PCR Assay Development Challenging?

The challenge is in understanding the capabilities of real-time PCR technology, and then treating it as any other scientific endeavor. Systematic step-by-step method, reaction, or design improvements should be the rule.
Often, because the vast number of reaction variables, assay development scientists choose to manipulate the ‘usual suspects’ for PCR: buffer, salts, temperatures. However, without a thorough grasp of the enzyme kinetics and hybridization kinetics involved in the PCR itself progress in assay development can stagnate. Then due to the ‘lack of progress’ or “new corporate priorities”, the project can be dropped and the real-time PCR machine gathers dust on the same benchtop that once held the aforementioned promise.
Developing a real-time PCR assay to quantify, to genotype, or to simply detect a target can be efficient and effective. It only requires that a researcher establish clear project objectives to what achievable in their real-time PCR instrument(s).
According to Fluoresentric’s design team, however, even the simplest of goals often provoke frustrating questions that need to be answered at the start of any new project:

1. What is the end-goal of the assay? Including limits of sensitivity and specificity, what should the real-time PCR assay do?
2. What is possible with the real-time PCR instrument in your lab?
3. What is feasible within a real-time PCR format? Does real-time PCR as a technology make sense for the project?

Knowing your objective for real-time PCR:
When beginning an assay development scientists are often vague about a project even if they have a clear end-goal in mind. When beginning a new project the Fluoresentric assay design team asks “What do you want your real-time PCR assay to do?” A typical response might be: “I want a detection assay”, or “I need to quantify a target”. For some researchers the goal is a tremendous undertaking requiring systematic research and development with his or her real-time PCR machine to determine the feasibility of their request. Somewhere between the extremes of “I want to quantify this target” and “I want to quantify 25 targets using a single multiplex reaction with 6 different dye combinations”, is most likely the actual project objective. “Leading a researcher to clear assay specifications is the most vital ingredient to successful assay development. We shouldn’t try to produce an ‘egg-laying-whole-milk-producing pig’ when a pig will do.” states Dr. Caplin.
To decide what an assay should do requires at least one clear objective. The quantification of gene expression for a single target with an endogenous reference standard, or genotyping the allele combinations at codon 20 of your gene of interest (GOI) are suitable objectives to start an assay development. If your project goals include potentially incorporating a second assay, such as also genotyping codon 28 of your GOI, plan for that as well. It is prudent to plan and design the assay for as many desired possibilities at the outset.
The result of planning an assay early on, even if only one assay is used immediately, increases the efficiency of development for a second, third, or even fourth assay in a multiplex reaction. The development time for the first assay will not have been wasted because the validated first assay does not require redesign to make other targets possible. Additionally, your real-time PCR staff will not have to settle for less than acceptable secondary assays that were included in a multiplex reaction as afterthoughts to the original target assay.
Think about all of the project’s objectives and squeeze into the design of the project as much as is possible. In addition, be aware of assay limitations especially those limitations of the instrument(s) available for the project.

Knowing what is Possible with Your Instrument:
It isn’t possible to address the myriad of real-time PCR instrument options. All real-time PCR instruments do the following: heat, cool, detect, report. The technical specifications of how well YOUR instrument heats, cools, and detects fluorescence can greatly impact the performance of an assay. The technical specifications for heating, cooling, and detecting will determine how robust (or flexible) the optimized assay needs to be to perform on YOUR real-time PCR instrument. The reporting and analysis features also impact the suitability of an instrument for a particular type of assay.
Some basic questions should be answered about your real-time PCR instrument(s) prior to assay development:

Heating (Cooling)

• How is the sample heated? (cooled?)
• Is it evenly heated in all samples? (cooled?)
• How fast does heating occur? (cooling?)
  Fluoresence Detection:
• What are the excitation wavelengths?
• What are the emission wavelengths?
• How many dyes can be used in a single reaction?
• How many fluorescence acquisitions per cycle?
• How is detection performed after the run is complete, can you do melt-curves?
• What are the resolution capabilities of the instrument during a melt?

Reporting

• Does your instrument have an integrated data analysis package?
• Can analysis be performed in real-time?
• Are the types of data analysis necessary for the project available with the instrument?
  o If not is there a ‘third-party’ software package that is available?
  o Is the ‘third-party’ software updated regularly, and are upgrades compatible with upgrades to your instruments software?
  Consider these features carefully when purchasing a new instrument or when establishing the technical specifications for a new assay. Dr. Caplin suggests, “Spend a little more of your project dollars to get a real-time PCR machine that has all of the flexibility you need in the thermal cycling programming, the detection formats, AND in the data analysis.”.
  

Knowing What is Feasible in a Real-time PCR Format:
“At Fluoresentric, we encounter inquiries about developing a real-time PCR assay to detect as many as 1500 different targets on 10 samples each.” Certainly such an undertaking is possible, though the best method may not be the use of real-time PCR. Such a project may be better suited for a conventional PCR, where specific probes are not involved and the cost of performing the individual reactions can be lower.
Another common inquiry: “Can SYBR Green I [a fluorescent double stranded DNA binding dye] based approach be used in lieu of probes?” The answer is quite simply yes, though the design and optimization of such reactions to produce clean results without the commensurate non-specific products (which also produce nice signals with SYBR Green I) is more time consuming than perhaps is warranted for such projects.
Real-time PCR is a terrific tool for those scientists who are planning to perform few assays (100 or less) on several hundred or even thousands of samples each. Projects such as genotyping allele variants (SNPs), disease predisposition, residual disease and especially DNA or RNA quantification (including gene expression, viral load, bacterial load) are ideal projects for real-time PCR.

The Promise Renewed: A Real-Time PCR Success Story
Fluoresentric recently encountered an exceptionally well prepared biopharma company who, “needed to detect the presence of the genomic DNA of a cell line that is used to grow our product, and the assay should quantify as little as 1/3000th of the cell’s genome.” Unfortunately, no sequence data for the cells being used was available in public or private databases.
“The project proceeded in two phases. Phase I began with an extensive search of closely related cell lines and genomic DNA sequences that could be helpful. Our design team uncovered several highly conserved sequences in the related cell lines, and began to design a basic detection assay to identify those which might be suitable for the customer’s cells,” says Dr. Caplin. The Fluoresentric design team recommended to the client’s scientists that they consider including a quantification reference standard that would allow them to normalize their samples for DNA loading and account for the presence of any PCR inhibitors. After several design iterations Fluoresentric had uncovered a suitable target for the drug producing cell, AND also one for the reference cell line.
The Fluoresentric design team then set out to develop a specific and sensitive real-time quantitative PCR assay, and Phase II began. The resulting assay was elegant in its simplicity. It was only necessary to use the client’s existing DNA standards to establish the sensitivity of the assay. In this project the biopharma R&D group had already established a concentration of DNA and since the assay was to be only a method to quantify the total DNA in the sample. Fluoresentric needed only to establish the limits of detection (LOD) and the limits of quantification (LOQ) of the assay.
“When the customer’s Quantitative PCR assay was confirmed for specificity and sensitivity, our Assay Development Team put the final touches on the assay, including a minor a probe redesign to produce stronger signals and improve sensitivity. The Assay Development Team put together a sample ‘kit’ of reactions with basic instructions for the customer and sent it off to be tested in their labs. Our clients were thrilled by the ease with which they could perform the assay themselves.”
“The last step was to transfer the assay out of the Fluoresentric lab. Some clients ask Fluoresentric to simply send the assay instructions, oligonucleotides, and templates so they can proceed with the assay. Others ask that we come to their site and teach them how the assay should be performed. For this project, a Fluoresentric Development Specialist went to our customer’s site and over the course of 2 days performed the quantitative real-time PCR assay side-by-side with the client’s laboratory specialists. The scientists there had the opportunity work with the assay with Fluoresentric assisting at every step.”
The assay that has been described (though vaguely) was developed over 1 year ago and the customer has now fully implemented this assay into their ‘in process’ quality program. During this time Fluoresentric has consulted with the customer on several occasions at no charge to the customer.
Dr. Caplin promises, “Every assay we develop comes with sufficient post-delivery consultation time to ensure the client’s questions are addressed without any additional expense to them. The client will have the assay they want, and it will be running in their lab.” The promise of real-time PCR technology is renewed, and Fluoresentric is here to keep it.
Fluoresentric is an unconventional real-time PCR kit company. Fluoresentric does not develop ‘generic’ real-time PCR assays to suit everyone’s needs. The design team at Fluoresentric customizes real-time PCR assays to meet individual user needs…to literally provide our clients a kit for the assay of their choice, designed to their specifications, and tested with their samples…Custom real-time PCR kits. On the day of assay delivery Fluoresentric’s assay development specialist’s goal is to deliver the client’s custom real-time PCR assay that performs exactly as intended, and if possible even exceed the client’s expectations.

How to Find Us:
Our website is filled with information and product offerings that are suitable for the real-time PCR and conventional PCR community:.
Visit us online at www.fluoresentric.com, email us (info@fluoresentric.com), or call us toll free (800 808 0490) and find out what Fluoresentric can do for your PCR.