Real-Time RT-qPCR

Ultra-Accurate & Hyper-Sensitive Real-Time RT-qPCR Assays

Table Of Content
1. The Need For A Bioanalytical Assay With Superior Selectivity.
2. Bioanalytical Real-Time RT-qPCR Assay Development Protocols.
3. One Quick Word to siRNA-Mediated Liver mRNA Knockdown Duration.
4. Almost No Influence of Small RNA 2´-O-Methyl Modifications.
5. References.


1. The Need For A Bioanalytical Assay With Superior Selectivity.

“If you perform these assays in 10 µl final volume, in a 384 well plate format, and on a rapid Real-Time qPCR cycler, such as the Applied Biosystem´s ABI 7900HT Fast Dx system, the costs per sample analysis could be kept low.
Even if you include 30 NTC samples (no template controls), 8 standards, 4 QCs, and (for tissue samples) an endogenous small RNA control; and all of these are assayed in triplicate, you can assay more than 60 triplicate samples onto the same plate at once.”

This is a statement I´m expecting to appear on the siRNA community´s bulletin board every day. It´s only a question of time. Although conventional dual-probe/capture probe/sandwich hybridization assays (or whatever these assays are termed) are reliable, robust, and FDA approved, the last years revealed the need for an ultra-sensitive quantification method as the in-life drug doses decrease. Also, the method should enable to quantify both the guide and the passenger strand, because one of these are expected to exhibit a prolonged elimination half-life over the other – given that RNA interference (RNAi) occurs in vivo.

So let´s get closer to the hands-on experimental steps of the outstanding Real-Time RT-qPCR assay. There are two main problems one has to tackle with: 1. the analyte siRNA is double-stranded, and (2) the elimination of the RNA purification step to compete against the (less sensitive, I must admit) sandwich hybridization assays. The short history of Real-Time microRNA (miRNA) or siRNA quantification could be illustrated as:
1. Quantification of single-stranded microRNA (miRNA) by 2-step RT-qPCR.
2. Introduction of pulsed RT to increase sensitivity for single-stranded microRNA.
3. Quantification of double-stranded short interfering RNA (siRNA) by 2-step RT-qPCR.
4. Competitive siRNA quantification (Ct value increases along with increasing siRNA conc).
5. Advancement of Real-Time RT-qPCR towards bioanalytical method development and validation (matrix effects).

Certainly these milestones in bioanalytical Real-Time RT-qPCR assay development and validation are commercially driven. As the doses decrease, the classic sandwich hybridization ELISA fall out of assay range (below limit of detection, LOD). As we all know, doses go down basically because, 1. liposomal shuttles can trigger an unrequested immune response (liver tox, complement activation, etc [Ref. [6], Figure 4]), 2. off-target effects sometimes are dominant over the on-target (if there´s any, see also Ref. [15]), and 3. CMC costs can be kept low.

To address the strong need for a sensitive bioanalytical quantification method for siRNA therapeutics from in-study samples, Merck & Co and its subsidiary Sirna Therapeutics (merger with Merck & Co in 2006) spent extraordinary time in the Real-Time RT-qPCR siRNA assay development. Recent excellent bioanalytical research on assay development and validation is summarized below.

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