For RNAi, direct interaction with a gene is not necessary to down-regulate gene functions. RNAi leads to the degradation of mRNA.
The effect of RNAi is proportional to the messenger RNA level and the translated protein level.
The down-regulation of gene expression can be determined as follows:
[1] Determine the activity of the target protein or enzyme. This option needs a robust assay. However, assays can vary due to the nature of the protein.
[2] Determine the level of the target mRNA. This approach directly quantifies mRNA levels. Quantitative PCR (qPCR) allows quantifying the relative amount of target sequences. qPCR assays utilize SYBR Green, TaqMan, or Molecular Beacons.