Can I use the same protocol for the transfection of siRNA and esiRNA?
The transfection conditions for siRNA and esiRNAs are comparable but not the same. Therefore one needs to optimize the conditions for esiRNAs separately. The conditions for siRNA transfection may serve as a starting point for this optimization.
Is my esiRNA targeting all transcript versions of the target-gene?
Yes, the esiRNAs are designed to target a region that is in common for all transcript versions of a gene. However, in rare cases a splicing variant is significantly different to all others so that no common region of sufficient length can be found. In those cases two esiRNAs are offered: one that targets the transcripts that shares a common region and another one that targets the version not covered with the first esiRNA.
How can I validate an observed RNAi phenotype?
RNAi phenotypes can be validated utilizing secondary esiRNAs that target a different transcript region of the primary esiRNA gene sequence. EsiRNAs for this purpose are available as “esiSEC” products. All esiSEC esiRNA are designed to be independent RNAi-triggers to our primary esiRNAs and are optimized by the DEQOR algorithm for specificity and efficacy.
What can be done if the knockdown is not strong enough?
Many reasons can lead to an unsatisfactory depletion of the target protein. Given that the transfection efficiency was optimized and is of good quality (please also refer to the section “Transfection of esiRNAs” for further details) the knockdown kinetics play an important role. For proteins that have a slow turn-over rate the maximum depletion can be expected 96 hours post-transfection (e.g. for the APC subunit Cdc27). If the utilized cell line divides slowly even a longer knockdown kinetics can be observed. If the turn-over rate is high the protein may deplete faster (e.g. for the motor protein Eg5) reaching the maximum after 48 hours. Eg5 is an essential protein and its depletion leads to massive induction of cell death. Therefore the cells that show significant protein depletion will escape measurements at a later time point duping a worse depletion rate in a Western Blot setup. However, in some cases it is worthwhile to try a secondary independent esiRNA (available as “esiSEC”) in order to target another region in the mRNA.
What can be done if my favorite gene is not in the list of esiRNAs?
Sigma offers silencing triggers for genes that are expressed. We are constantly adding new esiRNAs based on expression validation and updated genome annotations. For transcripts that are not covered, we offer custom-made esiRNAs (esiOPEN) that are fully flexible in terms of target-sequence and species. Here, DEQOR-optimized esiRNAs can be provided for any given transcript sequence (minimum length requirement 500 bases in length).
Are there esiRNAs available for other species than human or mouse?
Yes, Sigma offers custom-made esiRNAs (esiOPEN) that are fully independent of the origin of the sequences.
Are there esiRNAs available that target individual transcript versions of the same gene independently?
Yes, a custom-made esiRNA (esiOPEN) can be purchased for any target-region. Here the restriction is that a minimum length of 500 bases is required. Hence, the splicing version for targeting has to be different from the other splicing versions by at least these 500 bases in order to have specificity.
Transfection of esiRNAs:
Cell lines have different properties and are sensitive or insensitive to different manipulations. This is certainly also true for transfections. Therefore for every cell line an appropriate transfection protocol has to be established separately. The goal of transfection optimization is to achieve maximal transfection efficiency with minimal toxicity/cellular stress. Please note that the transfection conditions of chemically-derived siRNAs and esiRNAs may not be identical. However, known siRNA conditions can be used as a starting point for the optimization of esiRNA transfection.
Provided below are the esiRNA transfection conditions for some commonly used cell lines. The values provided should only serve as guidelines and a detailed optimization should be carried out if other plate formats are applied. We recommend using an esiRNA for Eg5 (Kif11) and Renilla luciferase (RLUC) as positive and negative controls, respectively for optimization. Eg5 (Kif11) induces a mitotic arrest with the formation of typically round-shape cells that will become apoptotic after some hours of arrest. These round shaped cells can easily be observed in a standard bright-field cell-culture microscope. The conditions with a maximum number of round-shaped cells for the Eg5 transfection with a minimum of toxicity for the RLUC transfection signify optimal transfection conditions.
esiRNA transfection conditions for various cell lines and plate formats