This review systematically covers sequence-specific gene regulation by miRNAs and dsRNA-derived siRNAs in animals and plants from the perspective of target RNA recognition, potential for non-target (off-target) effects, and reliable determination of biological effects of small RNAs in animals and plants. I will review sequence complementarity between target RNA and small RNA (siRNA or miRNA), including tolerance to mismatches, parameters influencing sequence complementarity (and target recognition and repression) and discuss specificity of targeting by miRNAs and off-targeting by siRNAs. In addition, I will discuss reliable identification of target RNAs (and, eventually, biological effects). Accordingly, the text is divided into the following four sections: (I) Small RNA:target RNA base pairing, (II) Other key factors influencing target recognition and repression, (III) Off-targeting -causes and remedies, (III) Smal RNA target identification.