Q1: Can I use the same protocol for the transfection of siRNA and esiRNA?
Answer: 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.
Q2. How many screenings can be performed with each of the 1.0 µg, 2.5 µg and 5.0 µg offerings of an esiRNA library?
Answer: Depending on your cell type, you will need 9 to 60 ng of esiRNA per well (384-well plate), or 30 to 200 ng of esiRNA per well (96-well plate). Assuming a 90% success rate, you can perform the following number of transfections:
| esiRNA (µg) | No. of transfections in a 384-well plate | No. of transfections in a 96-well plate |
|---|---|---|
| 1.0 | 15-100 | 4-30 |
| 2.5 | 37-250 | 11-75 |
| 5.0 | 75-500 | 22-150 |
Q3. Is my esiRNA targeting all transcript versions of the target-gene?
Answer: Yes, usually the esiRNAs are designed in a way that they 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.
Q4. How can I validate an observed RNAi phenotype?
Answer: RNAi phenotypes can be validated utilizing secondary esiRNAs that target another transcript region one of the gene of interest. esiRNAs for this purpose are available from Eupheria under the brand name “esiSEC”. All esiSEC esiRNAs are designed to be independent RNAi triggers to our primary esiRNAs and are optimized by the DEQOR algorithm for specificity and efficacy.
Q5. What can be done if the knockdown is not strong enough?
Answer: 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.
Q6. What can be done if my favorite gene is not in the list of esiRNAs?
Answer: Eupheria only offers silencing triggers for genes that are actually 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 purchased for any given transcript sequence (minimum length requirement 500 bases in length).
Q7. Are there esiRNAs available for other species than human or mouse?
Answer: Yes, Eupheria offers custom-made esiRNAs (esiOPEN) that are fully independent of the origin of the sequences.
Q8. Are there esiRNAs available that target individual transcript versions of the same gene independently?
Answer: 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 confer specificity.