Tools, Reagents, and Educational Resources
Several different RNAi strategies are currently used in gene knockdown experiments:
- Standard siRNAs consist of a 21−23 bp RNA duplex with a 2 nt overhang on the 3’ end of each strand
- Dicer-substrate siRNAs (DsiRNAs) are 25−27 bp RNA duplexes with a 2 nt overhang on the 3’ end of only the antisense strand
- Short-hairpin RNAs (shRNAs) are expressed by a vector in the nucleus, and then transported to the cytoplasm where they are processed and enter RISC as 21 bp dsRNAs
Each of these types of small RNAs is incorporated into nu¬cleoprotein RISC (RNA-induced silencing complex) via various intracellular mechanisms. Upon uploading, one strand of the duplex is released while the other (the antisense, or guide strand) is used to locate regions of complementarity in specific mRNA transcripts, resulting in mRNA cleavage and subsequent reduced gene expression from that transcript.
IDT offers design tools for all three types of RNAi molecules. These tools can be found on the website under the SciTools®
section. IDT recommends the use of DsiRNAs over standard siRNA duplexes as DsiRNAs are designed to be optimally processed by Dicer and show increased potency by engaging this natural processing pathway. Vector inserts for shRNA can be ordered as standard or Ultramer™ oligonucleotides—we recommend using Ultramer oligos. Ultramer oligos are high fidelity oligonucleotides synthesized with a high coupling efficiency, eliminating the need for purification. Use of Ultramer oligonucleotides may result in more effective cloning and quicker identification of the correct vector insert.
To access the free SciTools programs, or read more on Ultramer oligonucleotides, visit www.idtdna.com
. Also see the webinar, Planning and Executing siRNA Experiments
, on the IDT website or at www.youtube.com/idtdnabio
, for a review of good practices that drive optimal RNAi results. Author: Aftan Vander Zwaag, BS is a Technical Support Representative at IDT.