NEXT-RNAi prediction of reagent specificity

An RNAi reagent is said to be specific if it interferes with the intended target mRNA(s) only ('on-target') but with no other, unintended target mRNA(s) ('off-target').

NEXT-RNAi tries to optimize the reagent's specificity the following way:

NEXT-RNAi prediction of reagent efficiency

NEXT-RNAi enables the prediction of siRNA efficiencies according to two published sets of criteria:

Rational efficiency prediction

The 'rational' prediction includes 8 criteria to predict the efficiency of an siRNA (sense strand):

This scoring provides a score range from -2 to 10. In the studies done by Reynolds et al. a cut-off of 6 was used to define an efficient silencer (siRNAs with scores equal or higher than 6 are considered efficient).

For NEXT-RNAi the obtained scores are then normalized in the range of 0 – 100 using the following formula:

FinalScore = ((Score of siRNA - minimal possible score) / (maximal possible score - minimal possible score)) * 100

For an siRNA considered as efficient silencer this means:

FinalScore = ((6 + 2) / (10 + 2)) * 100 = 66.67

'Weighted' efficiency predicition

The 'weighted' prediction includes 12 criteria to predict the efficiency of an siRNA (sense strand):

This scoring provides a score range from -4.08 and 7.04.

For NEXT-RNAi the obtained scores are then normalized in the range of 0 – 100 using the following formula:

FinalScore = ((Score of siRNA - minimal possible score) / (maximal possible score - minimal possible score)) * 100

Shah et al. found in their study that the average score of the most potent siRNAs was 63.

Assessing regions of low sequence complexity

NEXT-RNAi avoids sequence-regions of low complexity (regarding base composition) from the RNAi reagent. Those were associated with 'off-target' effects and cytotoxic effects. Two filters are applied in the design process:

Standardized primer designs

NEXT-RNAi uses primer3 for the design of primer pairs required to amplify templates for long dsRNAs by PCR. All primer design parameters can be adjusted by the user, such as melting temperature, GC content, primer pair penalty and desired amplicon length. These settings are applied to all RNAi reagents designed in a single run, which facilitates efficient (high-throughput) PCR of all templates. It is recommended to use the same primer design settings for all reagents that are going to be synthesized side by side.

Design of independent RNAi reagents with NEXT-RNAi

E-RNAi provides two ways to design independent RNAi reagents: