A rapid and sensitive method to detect siRNA-mediated mRNA cleavage in vivo using 5' RACE and a molecular beacon probe

Abstract

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. 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. 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. 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%. 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.

Figure 1.

Comparison of 5′-RLM-RACE and MBRACE methods. The initial steps are the same in both methods, in which an RNA linker (striped bar) is attached to siRNA-cleaved mRNA (solid line). After cDNA synthesis by reverse transcription, a first round PCR reaction is set up using one primer specific for the linker sequence and a second primer specific to the gene target and product generated (dotted line). For standard 5′RACE (left), a second round PCR is performed using internal primers (Nested). Products are then analyzed by agarose gel electrophoresis, and amplicons of the predicted size are excised, cloned and sequenced to confirm that the product is derived from siRNA-mediated cleavage. For MBRACE, the first round PCR products are also used as template for a second round with the same nested primers, but the addition of a molecular beacon spanning the junction between the linker and cleaved mRNA enables amplification to be detected in real-time using a LightCycler® 480.

Figure 2.

Specific detection of mRNA cleavage products with MBRACE in vitro. (A) Schematic representation of sequences of: (1) the RNA linker (blue); (2) ApoB cleavage site; (3) positive (RNA linker ligated to cleavage product) and (4) negative (4 bp insertion between linker and mRNA fragment underlined) clones; and (5) the molecular beacon probe used to detect linker ligation to the cleaved mRNA fragment (red text represents the 7-bp stem). (B) Amplification curves showing fluorescent signal generated from positive (filled square) and negative (open square) cDNA clones. (C) RT–qPCR analysis of ApoB mRNA in Hepa1-6 cells transfected with either 1 (a) or 10nM (b) ApoB-1 siRNA, or 1 (c) or 10 nM (d) mismatch control siRNA. (D) The same samples were used as template in standard 5′-RLM-RACE analysis, and analyzed by electrophoresis. The correct product indicated by the arrow has a size of 290 bp. (E) Amplification curves from MBRACE reactions using the template from siRNA-treated cells as in (C) filled symbols represent samples transfected with ApoB-1 siRNA, and open symbols are samples transfected with mismatch control siRNA at 10nM (open square) or 1nM (open triangle). All MBRACE experiments were performed in duplicate, but for clarity only a single, representative replicate is shown.

Figure 3.

Application of MBRACE assay to other targets in vitro. MBRACE assays of RNA extracted from A549 cells transfected with (A) 1 nM and 100 nM of RRM1-2, (B) 1 nM and 100 nM of RRM1-3, (C) 10 nM YBX1-9 or (D) 10 nM RRM1-15. Filled symbols are samples transfected with RRM1 or YBX1 siRNAs; open symbols are from cells only control samples. YBX1 and RRM1 templates in (C) and (D) were tested with specific molecular beacon primers and probes (filled square) and also tested with molecular beacon primers and probes specific for RRM1-15 (filled triangle) and YBX1-9, (filled triangle), respectively. Inset graphs show siRNA-mediated mRNA knockdown using the same siRNAs as measured by RT–qPCR.

Figure 4.

ApoB knockdown and detection of mRNA cleavage products in mouse liver. (A) qRT–PCR of ApoB mRNA levels from the livers of mice treated with ApoB-1 siRNA or a mis-matched control siRNA by the HTVI method. Levels of ApoB mRNA were normalised to the average of the mis-match siRNA samples. (B) Standard 5′-RLM-RACE of the same samples, analyzed by electrophoresis on a 3% TAE-agarose gel using 1 kb Plus ladder (Invitrogen). An in vitro positive control (+ve) was prepared and analyzed in parallel; the band of correct size (290 bp) is indicated with an arrow.

Figure 5.

Detection of ApoB mRNA cleavage in vivo with MBRACE. Detection of ApoB knockdown in vivo using the MBRACE method was tested in three independent HTVI experiments, (A–C). Top panels show RT–qPCR analysis of ApoB expression in animals treated with the ApoB-specific siRNA ApoB-1 (A1–A7 in experiment A and A1–A6 in experiments B and C) or mismatch control siRNA (M1–M3). Amplification curves from the same samples were used for MBRACE reactions and fluorescence from the individual templates is shown in the middle panels, with average fluorescence from all specific (A) or mismatch control (M) siRNA-treated animals shown in the bottom panels. All individual MBRACE experiments were performed in duplicate but for clarity only single replicates are shown.