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

- 'Rational' prediction according to Reynolds et al.
- 'Weighted' prediction according to Shah et al.

### Rational efficiency prediction

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

- G/C content (+1 if between 30% and 52%)
- A/T bases at positions 15-19 (+1 each)
- Absence of internal repeats (+1 if melting temperature of potential internal hairpin < 20°C)
- A base at position 19 (+1)
- A base at position 3 (+1)
- T base at position 10 (+1)
- G/C at position 19 (-1)
- G at position 13 (-1)

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 E-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):

- A/T base at position 1 (-1.4)
- G/C base at position 1 (+1.11)
- A base at position 6 (+0.7)
- T base at position 10 (+0.25)
- G base at position 13 (-1.66)
- T base at position 13 (+0.31)
- A/T base at position 16 (+0.74)
- A/T base at position 17 (+1.2)
- A/T base at position 18 (+1.44)
- A/T base at position 19 (+0.87)
- G/C base at position 19 (-1.02)
- G/C content (+0.42 if between 30% and 55%)

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

For E-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.