Northern blotting analysis of microRNAs, their precursors and RNA interference triggers
© Koscianska et al; licensee BioMed Central Ltd. 2011
Received: 7 January 2011
Accepted: 11 April 2011
Published: 11 April 2011
Numerous microRNAs (miRNAs) have heterogeneous ends resulting from imprecise cleavages by processing nucleases and from various non-templated nucleotide additions. The scale of miRNA end-heterogeneity is best shown by deep sequencing data revealing not only the major miRNA variants but also those that occur in only minute amounts and are unlikely to be of functional importance. All RNA interference (RNAi) technology reagents that are expressed and processed in cells are also exposed to the same machinery generating end-heterogeneity of the released short interfering RNAs (siRNAs) or miRNA mimetics.
In this study we have analyzed endogenous and exogenous RNAs in the range of 20-70 nt by high-resolution northern blotting. We have validated the results obtained with northern blotting by comparing them with data derived from miRNA deep sequencing; therefore we have demonstrated the usefulness of the northern blotting technique in the investigation of miRNA biogenesis, as well as in the characterization of RNAi technology reagents.
The conventional northern blotting enhanced to high resolution may be a useful adjunct to other miRNA discovery, detection and characterization methods. It provides quantitative data on distribution of major length variants of abundant endogenous miRNAs, as well as on length heterogeneity of RNAi technology reagents expressed in cells.
MicroRNAs (miRNAs) are endogenous short RNAs (~22 nt) that control gene expression at the posttranscriptional level. There is growing evidence that miRNAs regulate various physiological processes and are frequently misregulated in many diseases [1–9]. The biogenesis of animal miRNAs includes two RNA cleavage steps (reviewed in [10–13]). First, in the nucleus, primary miRNA transcripts (pri-miRNA) are cleaved into approximately 60 nucleotide-long pre-miRNA precursors by the ribonuclease Drosha acting together with DGCR8 protein within the complex named Microprocessor [14, 15]. Then, the pre-miRNAs are exported to the cytoplasm by Exportin-5 [16, 17] and cleaved further by the ribonuclease Dicer protein complex into ~20 nucleotide-long miRNA duplexes [18, 19]. One of the two RNA strands becomes functional miRNA via Argonaute protein binding, and the other is released and degraded [20, 21]. Mature miRNAs are heterogeneous in length, varying between 19 and 25 nt [22–25]. The primary source of miRNA length heterogeneity is imprecise cleavage by the ribonucleases Drosha and Dicer . Further, miRNA 5'-end selection occurs upon Argonaute protein binding . The miRNAs that differ in their 5'-ends have different seed sequences and may regulate different sets of targets [24, 28–30]. Detection of the cellular levels of individual length variants of miRNAs with high precision is therefore very important. Similarly, determination of the exact length distribution of reagents released from the vectors used in RNAi and miRNA technologies is of importance because it may influence their performance in cells . It is also advantageous to monitor the lengths of reagents released from the vectors with regard to the off-target effects that these products may cause [32, 33].
Numerous reports have described various improvements of the northern blotting technique [34–39]. In this study, we use the method refined for extremely high-resolution detection of miRNAs, pre-miRNAs, siRNAs released from vectors, and any short RNAs of corresponding lengths. We demonstrate the usefulness of this northern blotting procedure by showing examples of its application in miRNA and RNAi fields to evaluate the precision of Drosha and Dicer cleavages.
Results and discussion
We show here that northern blotting of short RNAs that are 20-70 nt in length may provide insightful information on the distribution of individual length variants of siRNAs, miRNAs and their precursors in cells. We first show that high-resolution northern blotting and deep sequencing give similar results for abundant miRNAs. Then, we advance our recent observations showing utility of this northern blotting protocol for evaluating precision of Drosha and Dicer cleavages during human miRNA biogenesis . Finally, we put special emphasis on the need for better characterization of reagents released from expression constructs used to activate RNAi in cells.
Correlation between high-resolution northern blotting and deep sequencing results
Application of northern blotting in studies of miRNA and pre-miRNA length heterogeneity
High-resolution northern blotting in siRNA studies
In this study we have shown that the optimized high-resolution northern blotting can be used to analyze endogenous and exogenous RNAs in the range of 20-70 nt. We demonstrated the usefulness of this technique in the investigation of miRNA biogenesis as well as in the characterization of RNAi technology reagents. We have validated the high-resolution northern blotting as a reliable tool for length heterogeneity analysis of miRNAs and their precursors and have presented examples of its application in miRNA and siRNA studies. The method can be used in a variety of applications to verify mechanisms of RNAi-mediated effects. However, we understand that there is a limit to the interpretation of northern blots, and other techniques have to be used to provide complementing information. The techniques allowing precise mapping of both 5' and 3' ends of processed products include deep sequencing, primer extension (5') and rapid amplification of cDNA ends (5' and 3' RACE). These methods, when used jointly, will provide more complete and more reliable information about the exact lengths and end-sequences of miRNA and cell-expressed siRNA variants. Such information is very important as miRNA and siRNA variants having different 5' ends may differ in the potency to activate RISC [27, 49], as well as in downstream silencing effects. RISC programmed by different miRNA 5'-end variants may regulate different targets [24, 28–30], and programmed by siRNA variants may cleave mRNAs at shifted sites compromising the allele-specific SNP-targeting applications.
The animals were kept under standard conditions with a 12-h light/dark cycle and water and food ad libitum. The animals were sacrificed by placing them in a 70% CO2 atmosphere. The original strains C57BL/6J and C3H/HeJ were obtained from The Jackson Laboratory (Bar Harbor, Maine; USA) and were bred to B6C3F1.
The study was carried out in strict accordance with Polish Law on Animal Experimentation which complies with EU standards. All procedures and animal handling were carried out to minimize animal stress and were approved and monitored by The Local Ethical Commission for Animal Experiments in Poznan (Decision Number: 49/2010).
HEK 293T cells were obtained from the American Type Culture Collection (ATCC) and grown in Dulbecco's Modified Eagle's Medium (DMEM, Lonza) with 10% fetal bovine serum (FBS, Sigma-Aldrich) and Antibiotic Antimycotic Solution (Sigma-Aldrich) at 37°C in a humidified atmosphere of 5% CO2.
HEK 293T cells were grown to 90% confluence in T-25 flasks and transfected with 3 μg of either plasmid constructs (System Biosciences) encoding appropriate miRNA precursors or plasmid vectors (pSilencer 3.1-H.1 hygro, Ambion) containing specific expression cassettes (shRNA) (Additional file 1: Supplemental Table S2), using Lipofectamine 2000 (Invitrogen). The cells were harvested 24 hours after transfection, and isolated RNAs were analyzed by northern blotting.
RNA isolation and northern blotting of miRNAs and pre-miRNAs
Total RNA was extracted from the cells and selected mouse brain and muscle tissues using TRI Reagent (MRC, Inc., BioShop) according to the manufacturer's instructions. RNAs (20-30 μg) were resolved on denaturing polyacrylamide gels (12% PAA, 19:1 acrylamide/bis, 7 M urea) in 0.5 × TBE. Two separate electrophoresis runs were performed, as described previously [26, 50]. Briefly, a vertical electrophoresis gel system (II xi Cell, BioRad) for resolution of miRNAs, and a model S2 sequencing gel electrophoresis apparatus (Gibco, Life Technologies) for pre-miRNA separations were used. Xylene cyanol dye (XC) migrated 10 cm and 30 cm, for high resolution of miRNA and pre-miRNA fractions, respectively. Marker lanes contained a mixture of simultaneously radiolabeled synthetic RNA oligonucleotides (ORNs: 17-, 19-, 21-, 23-, 25-nt or ORNs: 60-, 61-, 63-, 64-nt) or RNA Low Molecular Weight Marker (USB). RNAs were transferred to GeneScreen Plus hybridization membrane (PerkinElmer) using semi-dry electroblotting (Sigma-Aldrich), immobilized by subsequent UV irradiation (120 mJ/cm2) (UVP) and baked in an oven at 80°C for 30 min. The membranes were probed with specific oligodeoxynucleotides (ODNs) complementary to the annotated mouse miRNAs miR-9, -9*, -93, -29a, -29b, -124, -132, -137, -191, -496, -1 and -206 (miRBase) and to 21-nt siRNAs generated from shRNAs (Additional file 1: Supplemental Table S3). The ODNs were labeled with [γ32P] ATP (5000 Ci/mmol, Hartmann Analytics) using OptiKinase (USB) according to the manufacturer's instructions. Pre-hybridizations and hybridizations were carried out under the same conditions at 37°C overnight in buffer containing 5 × SSC, 1% SDS and 1 × Denhardt's solution. After hybridization, the membranes were washed three times in a low-stringency buffer solution (2 × SSC and 0.1% SDS) for 20 minutes. Radioactive signals were quantified by phosphorimaging (Multi Gauge v3.0, Fujifilm).
We would like to thank Maciej Figiel and Pawel Switonski for providing mouse tissues for miRNA expression analyses.
This work was supported by the Polish Ministry of Science and Higher Education (grant numbers N N301 523038 and N N301 284837) and by the European Regional Development Fund within the Innovative Economy Programme (POIG.01.03.01-30-098/08).
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