Extended Gene Silencing
Bio-Rad has collaborated with Integrated DNA Technologies to develop RNAi tool sets. The results of this two-year collaboration, which pairs IDT’s siRNA design and synthesis proficiency with Bio-Rad’s transfection and amplification expertise, delivers validated Dicer-Substrate 27-mer small interfering RNA (siRNA) duplexes for RNAi applications. “We’ll be presenting silencing longevity and potency data for Dicer-Substrate 27-mer siRNAs and matched 21-mer siRNAs,” reports Eli Hefner, Ph.D., senior scientist at Bio-Rad. “We will also be discussing our Dicer-Substrate 27-mer (siLentMer) product validation procedure,”
Dr. Hefner points out that not everyone has heard of synthetic Dicer-Substrates. Dicer-Substrate siRNAs (DsiRNAs), which have recently been employed for in vivo studies using intraperitoneal and intrathecal routes of administration, had their beginnings with John Rossi at City of Hope. In vivo, long dsRNAs are cleaved by the RNase III class endoribonuclease dicer into 21–23 base duplexes having 2-base 3´-overhangs. These species, called small interfering RNAs (siRNAs), enter the RNA-induced silencing complex and serve as a sequence-specific guide to target degradation of complementary mRNA species.
“Despite the continuous evolution of sequence selection algorithms, many synthetic siRNAs still fail to achieve the desired level of silencing,” adds Dr. Hefner. “The only way to truly know that your siRNA will work is to buy one that was pretested. Even when an siRNA is capable of reducing the target gene mRNA to low levels, the corresponding protein concentration might not be affected. This can occur when the window of effective siRNA operation is shorter than the protein half life, hence the desire to identify RNAi-inducing molecules such as the siLentMer Dicer-Substrates that have a longer window of operation.”
Bio-Rad’s siLentMer-validated siRNAs help solve this dilemma by delivering effective gene silencing for an extended period of time using low siRNA concentrations, the company reports. According to Dr. Hefner and senior product manager Christina Whitman, they are functionally tested via RT-qPCR for 85% mRNA knockdown, and many will demonstrate sustained silencing for up to 6–9 days.
“We validate by testing in-house, and we are stringent with our passing criteria,” says Whitman. “For some experiments, synthetic siRNAs are able to achieve 70 percent silencing, which might be okay depending on your protein stability. With the validated siLentMer Dicer-substrates you are guaranteed the highest possible silencing of 85 percent or higher.”