Beginning in late October / early November 2020, esiRNA products that are delivered in tubes — Individual esiRNA, esiOPEN, and esiSEC — will begin shipping lyophilized instead of in solution (esiRNA products in plates — esiLibrary and esiFLEX — will continue to ship in solution). We are making this change to products in tubes (most esiRNA ships in tubes) for the following reasons:
MISSION® esiRNA—powered by Eupheria Biotech—provide RNAi researchers with a proven, cost-effective, and simple way to perform post-transcriptional silencing of protein-coding genes and lncRNA (long non-coding RNA). Biologically-prepared, esiRNA are comprised of a heterogeneous pool of siRNA (natural RNA, no modifications) that all target the same mRNA sequence. These multiple silencing triggers lead to highly specific and effective gene knockdowns with lower off-target effects than single, chemically-synthesized siRNA (Figure 1).
Figure 1.Knockdown of target mRNA can be accomplished by chemically-synthesized siRNA or enzymatically-prepared siRNA (esiRNA). A) Chemically-synthesized siRNA consists of a single silencing trigger of 21 bp that is complementary to the target mRNA. The high concentration of the siRNA in the transfection reaction leads to pronounced off-target effects. B) In contrast, esiRNA consists of a pool of hundreds of siRNA (21 bp) that cover a region of 300 – 600 bp of the target mRNA. Each individual siRNA has a lower concentration in the pool, leading to lower off-target effects, while producing an efficient knockdown.
Figure 2.Evaluation of gene knockdown in HeLa cells transfected with esiRNA directed against Renilla Luciferase (negative control) and different expressed targets at mRNA and protein levels. A) qPCR data validate knockdowns 24 hours post-transfection. The mRNA abundance was reduced by >90% in all transfections. B) Quantitative Western Blot data validate efficient knockdown 72 hours post-transfection. Protein levels were reduced 45 to 90%.
The following apply to all product options presented in the esiRNA specifications table:
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A variety of positive and negative controls are available (Figure 3):
Figure 3.Phenotypic analysis of HeLa cells transfected with esiRNA against A) RLUC (negative control) and B) Eg5/KIF11 (positive control). RLUC does not induce any phenotypic changes, while Eg5/KIF11 induces mitotic arrest, shown by rounded cells.
Transcriptome analyses reveal that up to 90% of the genome is transcribed into non-coding RNA, which among other types, includes lncRNA. The lncRNA are associated with different functions, including chromatin modification, co-activation of transcription factors, transcription, interaction with RNA-binding proteins, and repression of promoters. In addition, lncRNA have been implicated in diseases such as cancer. The details of how lncRNA work must be further elucidated. The esiRNA are an effective screening tool to study lncRNA function (Figure 4).
Figure 4.qPCR data validating knockdown of lncRNA in HeLa cells 24 hours post-transfection with corresponding esiRNA. Controls were transfected with esiRNA against RLUC and used for normalization.
Biologically-prepared (Figure 5), esiRNA is manufactured by cleaving long dsRNA (double-stranded RNA) with the endoribonuclease, E. coli RNase III
Figure 5.Manufacturing overview of esiRNA for single genes. A) DEQOR is used to select the mRNA target region that will produce the largest number of highly-effective siRNA, cover all known transcript variants, and minimize off-target effects. B) The target region is extracted from the cDNA clone and amplified via PCR (both primers are used to introduce T7 promoters. C) The PCR product is transcribed in vitro utilizing RNA polymerases. D) The annealed and long, double-stranded RNA (dsRNA) is digested with RNase III, and purification removes the DNA template, residual NTPs, and incompletely digested dsRNA. Finally, esiRNA is ready as a pool of hundreds of siRNA with an average duplex length of 21 bp.
MISSION® esiRNA will reduce target mRNA levels by 70%, in cultured cells, when transfected at a concentration of ≥30 nM. If the esiRNA does not knock down the target gene by 70%, we will provide an additional esiRNA for that gene, free of charge. If there are no more esiRNA for that gene, we will refund the purchase price.
Receipt of appropriate supporting data for transfection efficiency is required for the guarantee. Appropriate supporting data for transfection efficiency would include qPCR data comparing target mRNA levels of a validated MISSION® esiRNA, transfected at ≥30 nM, to an appropriate negative control (such as mock transfection, RLUC, FLUC, or eGFP esiRNA), demonstrating knockdown of the target mRNA of 70%.
Due to the variability of antibodies and protein half-lives, we are unable to accept data from protein-based detection methods.
To learn more about esiRNA, visit our Frequently Asked Questions section.
Learn about screening in Mammalian cells with esiRNA.