![]() ![]() In standard 5′-RLM-RACE, a dephosphorylation step is used to remove any RNAs that have a 5′phosphate (including nicked transcripts), effectively enriching for the capped full-length mRNA, which can then be de-capped and ligated. This may be because RLM-RACE appears to be an inefficient process for detecting mRNA cleavage products, despite the inherent specificity of using target and linker-specific primers. Although RACE-based detection has been utilised to assess the action of antisense-based inhibition of expression ( 18), it has only rarely been used in the corroboration of knockdown data observed in an ever increasing range of studies using RNAi in vivo ( 12–17). The amplified fragment must then be cloned and sequenced to ascertain the junction between the RNA linker sequence and the target RNA sequence which, if mRNA cleavage has occurred via RNAi, should correlate with the position as predicted by the siRNA sequence ( 12–17). ![]() Either of these fragments can be amplified using RACE in a modified version of the RLM-RACE protocol ( 10, 11). The slicing action of the RISC-associated Argonaute 2 protein at this position cleaves the target mRNA into two distinct fragments: a 5′ fragment with a 3′ hydroxyl group, and a 3′ fragment with a 5′ phosphate. More recently, it was demonstrated that siRNAs able to inhibit viral replication in vivo do so via stimulation of the innate immune system rather than through an RNAi-mediated event ( 7), and that the negative control used in these studies had an unusually low immunostimulatory activity, and had also been a component of several other in vivo studies assessing immunostimulatory siRNA.Ĭonfirmation that an observed mRNA knockdown has occured via an RNAi-based cleavage event can be demonstrated using the well-established technique of Rapid Amplification of cDNA Ends (RACE), as siRNAs cleave their target sequence following a canonical pattern 10 bp from the 5′-end of the antisense strand ( 8, 9). ( 6) demonstrated that the intravitreal injection of siRNA could lead to down regulation of vascular endothelial growth factor a (Vegfa) and its receptor (Vegfr1) in a sequence-independent manner, as siRNAs with no homology to Vegfa or Vegfr1 were as effective as specific siRNAs ( 6). The importance of confirming that mRNA knockdown following siRNA administration has occurred via an RNAi-mediated event is highlighted by two recent studies reporting the considerable contribution of the innate immune system to apparent in vivo knockdown, suggesting that many of the reports of in vivo efficacy of siRNAs can be explained by a general down-regulation of transcription that is stimulated by the double stranded RNA structure of siRNA without involving RNAi. siRNAs have the potential to trigger an innate immune response through the activation of Toll-like receptors (TLR 3, 7 and 8) and also by binding to proteins such as retinoic acid inducible gene 1, and this itself may cause a modulation in gene expression which could account for the observed effects attributed to siRNA-mediated RNAi ( 4, 5). Although many avenues of delivery are being investigated, including the use of localised delivery by direct injection and topical administration, and systemic delivery with intravenous administration ( 1–3) very few studies have included data to confirm that the effects of siRNA observed in vivo are due to an RNAi-mediated mRNA cleavage mechanism rather than non-specific events. The inhibition of gene expression by RNAi has great therapeutic potential but remains hampered by inefficient delivery of siRNA and potential off-target effects. With its sensitivity and specificity, this variation on the 5′RACE method should prove a useful tool to detect mRNA cleavage and corroborate knockdown studies following siRNA use in vivo. Detection of siRNA-mediated cleavage was also observed when RNA from mouse liver following administration of ApoB-specific siRNA was analysed, even in cases where ApoB knockdown measured by real-time PCR was <10%. When RNA from siRNA-transfected cells was used for 5′-RLM-RACE and a cleavage site-specific molecular beacon probe was included in subsequent real-time PCR analysis, the specific mRNA cleavage product was detected. We have combined 5′-RNA-linker-mediated RACE (5′-RLM-RACE) with real-time PCR using a molecular beacon to develop a rapid and specific method termed MBRACE, which we have used to detect small-interfering RNA (siRNA)-induced cleavage of ApoB, RRM1 and YBX1 transcripts in vitro, and ApoB in vivo. Specific detection of mRNA cleavage by 5′RACE is the only method to confirm the knockdown of mRNA by RNA interference, but is rarely reported for in vivo studies.
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