Custom Therapeutic siRNA Assays.
VERIFICATION OF CLEAVAGE SITE SPECIFICITY
A key concern in using siRNAs as human therapeutics is the demonstrated possibility for non-RNAi related off-target effects or even miss-targeting of mRNAs other than the expected transcript in some cases.
5’-RLM-RACE assay defines a site-specific, siRNA-directed cleavage. AltheaDx offers comprehensive custom therapeutic siRNA clinical services designed to verify the in vivo distribution of candidate siRNA molecules, to confirm target-specific mRNA cleavage by siRNA-mediated RNAi and to quantify the degree of knockdown.
RANGE OF APPLICATIONS
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Research and Development use: an assay validated and run in the spirit of GLP is appropriate for any study where the data is used to further research the drug’s performance |
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Investigational New Drug (IND) submissions: an assay validated and run in our GLP labs under GLP guidelines can produce data the can be submitted to the FDA as part of the regulatory filing to support IND submission |
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Clinical use: 5’-RACE assays can also be developed, validated and run in our CLIA certified laboratories so the data can be used for clinical treatment decisions |
PERFORMANCE PARAMETERS
| 1. |
Specificity |
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Validation of automated design, BLAST confirmation of sequence uniqueness, confirmation of ΔCt between samples, treated and untreated, in SYBR green (no probe) and TaqMan probe formats for RT-PCR |
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Qualitative gel images (to determine presence/absence of specific PCR product), oligo sequencing of all positive samples and alignment with the predicted mRNAGeneRacer oligo ligation product to confirm presence of the RNAi cut site using 5’-RACE assay |
| 2. |
Dynamic Range – Design and optimization of primer concentration and reaction conditions to bring output into optimal performance range using a representative set; 20-30 Ct for RT-PCR |
| 3. |
Reproducibility and Precision – Demonstration of assay performance in repeat study; 3 runs x 2 operators x triplicate run on more than one day |
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Intra-assay precision is demonstrated by the replicates within the same run |
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Inter-assay precision is demonstrated by different runs of same samples that account for the variability among different operators, different instruments, and different days |
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CVs to be no greater than 15% in cells and fresh frozen tissue derived, high quality RNA, no greater than 25% in FFPE tissue derived degraded RNA |
| 4. |
Linearity – Demonstration of signal linearity over a range of RNA concentrations |
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Four RT-PCR titrations are performed using test samples covering a 4 log titration (2 logs greater than and 2 logs less than the normal input RNA amount) |
