Amplifying the Value of qPCR & mRNA Analysis for Drug Discovery

How High Throughput qPCR and mRNA Analysis Are Being Used in Biopharma R&D

I imagine you are saying to yourself – the title was catchy, but is reading the whole article really worth it. Are you are interested in learning more about high throughput qPCR and mRNA analysis? Are you studying the downstream effects of a compound and would like to know more about the up regulation/down regulation of genes associated with a particular phenotype? Do you want to verify NGS results? If so, this is the article for you!

The New Genomic and Proteomic Era

New qPCR and mRNA analysis technology empowers scientists to run primary screens, perform secondary screening, analyze cellular pathways and confirm NGS/RNAseq results. Several engineering advances have revolutionized how we measure gene expression and the throughput with which we do so. Within the last few years, single well – single readout microliter qPCR has been transformed by advances in miniaturization (reduction to nanoliter volumes) and multiplex capability for investigation of differentially expressed genes in a single well.

Before we get started I want to ensure everyone is aware that this article is not a primer (no pun intended) on qPCR. High and low mRNA reference standards, dilution linearity, primer efficiency, spike and recovery…in case these words have been written over in your brain by more exciting facts, you may find it helpful to refresh with the MIQE Guidelines. Click here and be on the lookout for a future post going into more detail on this topic.

The Role of HT-qPCR in Drug Discovery

Before we get too far down the rabbit hole, let’s take a step back and discuss where qPCR fits into the drug discovery process.

We start with hundreds of thousands of compounds, which are made up from a diversity of chemical matter, and then apply compounds at single concentrations to physiologically relevant cellular systems or biochemical cross sections of a disease, and generate large sets of data.

Next, with data in hand, we work to validate if the compounds look more like our positive control or our negative control. Using hit criteria, compounds are evaluated to identify promising leads. Hit criteria can be as easy as defining a threshold three standard deviations above or below a control population, defining confidence intervals for univariate data, or using principal component analysis and dimension reduction for multivariate data sets.

After filters are applied to stratify hits for selection, ideally we arrive at a compound list not more than ~1% of compounds screened. In addition, we will have answered the question, “which control do our compounds look like?” with the goal of gating on compounds that ameliorate the disease process.

Next, through re-testing, dose responses, orthogonal screening, and counter screening, we weed out false positives-Type I Error (α) and false negatives- Type II Error (β). Now we have a compound list with multiple scaffolds to move on to the medicinal chemistry phase of drug discovery. And if all goes well, after increasing potency and efficacy, while limiting or reducing off target effects, we funnel worthy candidates into the pre-clinical phase.

Ideally, qPCR would be utilized during primary screening. Unfortunately, given qPCR’s low throughput, primary screening is an impossible task and qPCR is instead often deployed during the hit follow-up and/or confirmation phase. In fact, first generation qPCR and HTS (high throughput screening) were once considered mutually exclusive events because of the qPCR throughput barrier.

Advancements in Technology: HTS and qPCR are Mutually Inclusive

Up until recently HTS was a single readout (multiple targets were identified and were prioritized based on physiological relevance and assay robustness, and only one was advanced as a primary screening candidate) and screening for a genetic signature was what many scientists’ hopes and dreams were made of. But not anymore – qPCR now plays a pivotal role in multivariate HTS.


Through advancements in miniaturization, specifically, microfluidics to reduce well volume and the ability to multiplex reactions in a single well, HTS is now part of qPCR lexicon and should be a consideration in a drug discovery effort. These advancements are important not only because qPCR has transformed how we measure nucleic acids and is now an important step for expression validation generated by microarray analysis (including NGS and other genomics techniques) but it has also become a new technique for HTS.

Magnifying the Value of qPCR Advancement

Perhaps you are interested in 5, 10, or 96 differentially expressed genes as a surrogate endpoint for a phenotypic response. Or you are a protein biochemist interested in protein folding and studying up/down regulation of key molecular chaperons in protein folding pathways.

The ability to multiplex qPCR reactions empowers end-users to ask multiple questions per well in order to elucidate biological pathways. There are many great hardware high throughput systems available today each with their own advantages, which can multiplex qPCR reactions. Here is a partial list – Smart Chip (Wafergen Biosciences), BioMark HD (Fluidigm), OpenArray (Thermo Fisher Scientific), 1536 Light Cycler (Roche Life sciences) and HTG Edge for qNPA (HTG Molecular).

However, the hardware itself is not enough. To truly experience the value of the new process you must be able to manage the data. Thermo Fisher™ Platform for Science™ software lowers the activation energy required to record, analyze and collaborate in the proper scientific domains, empowering end users to seamlessly integrate powerful analytical tools and push biomedical research, drug discovery and science forward.

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