MicroRNA fate upon targeting with anti-miRNA oligonucleotides as revealed by an improved Northern-blot-based method for miRNA detection

被引:75
作者
Torres, Adrian G. [1 ]
Fabani, Martin M. [1 ]
Vigorito, Elena [2 ]
Gait, Michael J. [1 ]
机构
[1] MRC, Mol Biol Lab, Cambridge CB2 0QH, England
[2] Babraham Inst, Lab Lymphocyte Signalling & Dev, Cambridge CB22 3AT, England
基金
英国医学研究理事会;
关键词
microRNA; miR-122; oligonucleotide; Northern blot; RNA extraction; HEPATITIS-C VIRUS; LIVER-SPECIFIC MICRORNA; PEPTIDE NUCLEIC-ACIDS; ANTISENSE OLIGONUCLEOTIDES; IN-VIVO; MESSENGER-RNAS; POSTTRANSCRIPTIONAL REGULATION; SUBCELLULAR-LOCALIZATION; LNA PROBES; INHIBITION;
D O I
10.1261/rna.2533811
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
MicroRNAs (miRNAs) are small non-coding RNAs involved in fine-tuning of gene regulation. Antisense oligonucleotides (ONs) are promising tools as anti-miRNA (anti-miR) agents toward therapeutic applications and to uncover miRNA function. Such anti-miR ONs include 2'-O-methyl (OMe), cationic peptide nucleic acids like K-PNA-K3, and locked nucleic acid (LNA)-based anti-miRs such as LNA/DNA or LNA/OMe. Northern blotting is a widely used and robust technique to detect miRNAs. However, miRNA quantification in the presence of anti-miR ONs has proved to be challenging, due to detection artifacts, which has led to poor understanding of miRNA fate upon anti-miR binding. Here we show that anti-miR ON bound to miR-122 can prevent the miRNA from being properly precipitated into the purified RNA fraction using the standard RNA extraction protocol (TRI-Reagent), yielding an RNA extract that does not reflect the real cellular levels of the miRNA. An increase in the numbers of equivalents of isopropanol during the precipitation step leads to full recovery of the targeted miRNA back into the purified RNA extract. Following our improved protocol, we demonstrate by Northern blotting, in conjunction with a PNA decoy strategy and use of high denaturing PAGE, that high-affinity anti-miRs (K-PNA-K3, LNA/DNA, and LNA/OMe) sequester miR-122 without causing miRNA degradation, while miR-122 targeting with a lower-affinity anti-miR (OMe) seems to promote degradation of the miRNA. The technical issues explored in this work will have relevance for other hybridization-based techniques for miRNA quantification in the presence of anti-miR ONs.
引用
收藏
页码:933 / 943
页数:11
相关论文
共 54 条
[1]   Target RNA-Directed Trimming and Tailing of Small Silencing RNAs [J].
Ameres, Stefan L. ;
Horwich, Michael D. ;
Hung, Jui-Hung ;
Xu, Jia ;
Ghildiyal, Megha ;
Weng, Zhiping ;
Zamore, Phillip D. .
SCIENCE, 2010, 328 (5985) :1534-1539
[2]   Inhibition of HIV-1 Tat-dependent trans activation by steric block chimeric 2′-O-methyl/LNA oligoribonucleotides [J].
Arzumanov, A ;
Walsh, AP ;
Rajwanshi, VK ;
Kumar, R ;
Wengel, J ;
Gait, MJ .
BIOCHEMISTRY, 2001, 40 (48) :14645-14654
[3]   Antiviral activity of steric-block oligonucleotides targeting the HIV-1 trans-activation response and packaging signal stem-loop RNAs [J].
Brown, D ;
Arzumanov, AA ;
Turner, JJ ;
Stetsenko, DA ;
Lever, AML ;
Gait, MJ .
NUCLEOSIDES NUCLEOTIDES & NUCLEIC ACIDS, 2005, 24 (5-7) :393-396
[4]   MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells [J].
Chan, JA ;
Krichevsky, AM ;
Kosik, KS .
CANCER RESEARCH, 2005, 65 (14) :6029-6033
[5]   Liver-specific MicroRNA miR-122 enhances the replication of hepatitis C virus in nonhepatic cells [J].
Chang, Jinhong ;
Cruo, Ju-Tao ;
Jiang, Dong ;
Guo, Haitao ;
Taylor, John M. ;
Block, Timothy M. .
JOURNAL OF VIROLOGY, 2008, 82 (16) :8215-8223
[6]  
Chang Jinhong, 2004, RNA Biol, V1, P106, DOI 10.4161/rna.1.2.1066
[7]   Antisense inhibition of human miRNAs and indications for an involvement of miRNA in cell growth and apoptosis [J].
Cheng, AM ;
Byrom, MW ;
Shelton, J ;
Ford, LP .
NUCLEIC ACIDS RESEARCH, 2005, 33 (04) :1290-1297
[8]   The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on [J].
Chomczynski, Piotr ;
Sacchi, Nicoletta .
NATURE PROTOCOLS, 2006, 1 (02) :581-585
[9]   Trends in microRNA detection [J].
Cissell, Kyle A. ;
Deo, Sapna K. .
ANALYTICAL AND BIOANALYTICAL CHEMISTRY, 2009, 394 (04) :1109-1116
[10]   Improved targeting of miRNA with antisense oligonucleotides [J].
Davis, Scott ;
Lollo, Bridget ;
Freier, Susan ;
Esau, Christine .
NUCLEIC ACIDS RESEARCH, 2006, 34 (08) :2294-2304