How to handle your esiRNA

  • Please store the esiRNA at -20°C upon arrival. esiRNAs are stable under these conditions for at least 2 years.
  • Keep the esiRNA stocks at high concentrations. Avoid storing esiRNAs at concentrations less than 20 ng/µl for more than a few days. esiRNA concentrations of less than 20 ng/µl should only be prepared as working dilutions and are not suitable for storage.
  • Do not precipitate your esiRNA.
  • Dilutions should be done in TE buffer (10 mM Tris-HCl; 1 mM EDTA; pH 8.0).
  • esiRNA should always be kept on ice (4°C) during bench work.
  • Avoid multiple freeze/thaw cycles by aliquoting the stock.
  • If you work with plates: always make sure that the plates are completely thawed, vortexed and short spun before unsealing.
  • esiRNAs are not sensitive to vortexing. When esiRNAs are freshly thawed or diluted, make sure that they are rigorously vortexed before use.

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. 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 transfections.
Here, we provide the esiRNA transfection conditions as they were optimized in our laboratory 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 used. 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-shaped cells that will become apoptotic some hours after arrest. These round shaped cells can easily be observed in a standard bright-field cell-culture microscope. The conditions, which induce the maximum number of round-shaped cells after Eg5 transfection with minimum of toxicity after RLUC transfection, are optimal for your transfections.

esiRNA transfection conditions for various cell lines and plate formats

Cell line Origin Plate format Amount esiRNA [ng] Transfection reagent
R1/E 129Sv mouse ES 96-well 50 Lipofectamine 2000
E14Tg2a 129Sv mouse ES 384-well 20 Lipofectamine 2000
E14Tg2a 129Sv mouse ES 96-well 50 Lipofectamine 2000
E14Tg2a 129Sv mouse ES 6-well 500 Lipofectamine 2000
E14Tg2a 129Sv mouse ES 10-cm dish 7500 Lipofectamine 2000
SW48 Human colorectal carcinoma 96-well 40 Oligofectamine
DLD-1 Human colorectal carcinoma 96-well 50 Lipofectamine 2000
HCT116 Human colorectal carcinoma 96-well 50 Oligofectamine
HCT116 Human colorectal carcinoma 6-well 1200 Oligofectamine
RKO Human colorectal carcinoma 96-well 50 Oligofectamine
HeLa Human cervical carcinoma 384-well 15 Oligofectamine
HeLa Human cervical carcinoma 96-well 30 Oligofectamine
HeLa Human cervical carcinoma 6-well 1000 Oligofectamine
U2OS Human osteosarcoma 384-well 60 Oligofectamine, Effectene
U2OS Human osteosarcoma 96-well 30 Oligofectamine, Effectene
NIH3T3 Immortalized mouse embryonic fibroblasts 384-well 50 Lipofectamine, RNAiMAX
NIH3T3 Immortalized mouse embryonic fibroblasts 96-well 200 Lipofectamine, RNAiMAX
NIH3T3 Immortalized mouse embryonic fibroblasts 12-well 1600 Lipofectamine, RNAiMAX
HEK293 Human embryonic kidney 384-well 25 Effectene