A rapid and sensitive method to detect siRNA-mediated mRNA cleavage in vivo using 5' RACE and a molecular beacon probe.
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.
Lasham
Annette
A
Genesis Research and Development Corporation Limited, Auckland, New Zealand.
Herbert
Mike
M
Coppieters 't Wallant
Natacha
N
Patel
Rachna
R
Feng
Sheryl
S
Eszes
Marika
M
Cao
Helen
H
Reid
Glen
G
D000818:Animals; D001055:Apolipoproteins B / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D005260:Female; D005456:Fluorescent Dyes; D006801:Humans; D051379:Mice; D015345:Oligonucleotide Probes; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D001055:Apolipoproteins B; D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D034741:RNA, Small Interfering