Abstract
Background
microRNAs (miRNAs) are small single-stranded non-coding RNAs that act as crucial regulators of gene expression. Different methods have been developed for miRNA expression profiling in order to better understand gene regulation in normal and pathological conditions. miRNAs expression values obtained from large scale methodologies such as microarrays still need a validation step with alternative technologies.
Results
Here we have applied with an innovative approach, the Luminex® xMAP™ technology validate expression data of differentially expressed miRNAs obtained from high throughput arrays. We have developed a novel labeling system of small RNA molecules (below 200 nt), optimizing the sensitive cloning method for miRNAs, termed miRNA amplification profiling (mRAP). The Luminex expression patterns of three miRNAs (miR-23a, miR-27a and miR-199a) in seven different cell lines have been validated by TaqMan miRNA assay. In all cases, bead-based meas were confirmed by the data obtained by TaqMan and microarray technologies.
Conclusions
We demonstrate that the measure of individual miRNA by the bead-based method is feasible, high speed, sensitive and low cost. The Luminex® xMAP™ technology also provides flexibility, since the central reaction can be scaled up with additional miRNA capturing beads, allowing validation of many differentially expressed miRNAs obtained from microarrays in a single experiment. We propose this technology as an alternative method to qRT-PCR for validating miRNAs expression data obtained with high-throughput technologies.
Similar content being viewed by others
Background
MicroRNAs (miRNAs) are endogenous 18-24 nucleotides (nt) long noncoding RNAs (ncRNA) that control gene expression by targeting mRNAs and triggering either translation repression or degradation. The degree of complementarity between a miRNA and its mRNA target determines, at least in part, the regulatory mechanism [1]. Recently, a third less understood mechanism of small RNAs interference on gene expression involves heterochromatin silencing [2]. Many miRNAs are highly conserved among animals and plants [3] and it is estimated that up to 33% of all mRNA coding genes are negatively regulated by miRNAs [4, 2) shows that the xMAP™ technique is less expensive and more flexible allowing the simultaneous analysis of a larger number of miRNAs from the same sample. In addition, xMAP™ does not need for specific reverse transcription reactions for each miRNA and provides at least 100 independent measures for each miRNA, improving the statistical power. Moreover, xMAP™ expression data can be normalized respect to a spike fluorescence signal added in known quantities in the early stages of the labeling reaction, allowing the control over all stages of the reaction. In this way there is no need of an endogenous control, like in qRT-PCR, whose representativeness is sample-dependent.
Probably, a negative aspect of xMAP™ technology is represented by the use of enriched total RNA ( < 200 nt) that requires higher amounts of starting material respect to qRT-PCR. To avoid the enrichment step of < 200 nt RNA molecules, we propose the use of LNA capture-probe oligonucleotides that increase the affinity of the oligonucleotide for its complementary RNA target leading to a significant enhance in stability and specificity of the duplex.
We think that the technology we have developed could be an alternative method to qRT-PCR for validating miRNAs expression data obtained with a large scale technology such as microarray and it could have a wide application in clinical, pharmaceutical, agricultural and environmental studies.
Methods
Cell culture
Human alveolar RMS (ARMS) cell lines, positive for PAX3-FKHR translocation (RH4, RH30), negative for PAX3-FKHR translocation (RH18) and human embryonal RMS (ERMS) cells (RD, RH36, CCA, SMS-CTR) were maintained in modified Eagle's medium (DMEM) containing 10% fetal calf serum, penicillin (100 U/ml), and streptomycin (100 μg/ml) (Invitrogen) at 37°C, 5% CO2 in a humidified incubator.
The human RMS cell lines RH30 and RD were purchased from ATCC (Manassas, VA); RH4 and RH18 were a gift of Dr P.J. Houghton (St Jude Children's Hospital, Memphis, TN); SMS-CTR, RH36, CCA were obtained from Dr M. Tsokos (NCI, Bethesda, MD).
Isolation of small RNA molecules
Total RNA was prepared from seven cell lines of human rhabdomyosarcoma using a modified TRIzol (Life Technologies Corporation, Carlsbad, CA, USA) protocol for small RNA enrichment. A pellet of about 6 × 106 - 9 × 106 cells was dissolved in 1 ml of TRIzol and the supernatant, containing total RNA, was purified by PureLink™ miRNA Isolation Kit (Life Technologies Corporation) that was specifically designed to enrich total RNA preparation for < 200-nt RNA molecules. RNA quantity and quality were assessed by Nanodrop (NanoDrop Technologies, Wilmington, DE, USA) spectrophotometry and microelectrophoresis using Small RNA Nano LabChip by Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) respectively.
miRNA expression profiling and statistical analysis of data
microRNA expression profiling was carried out using the "mirVana Probe Set V1" (Ambion) that is a collection of about 400 amine-modified DNA oligonucleotides representing a panel of the human, mouse and rat microRNAome in the miRNA Registry (miRBase - Release 9). The probes are 42-46 nucleotides (nt) long, with 18-24 nt segment targeting a specific miRNA, and the remaining sequence serving as spacer. We analyzed the expression profiles of 7 different rhabdomyosarcoma cell lines: 3 ARMS (RH4, RH30, RH18) and 4 ERMS (RD, RH36, CCA, SMS-CTR). The miRNA population from each cell line was compared to a reference sample consisting of a pool of the 7 total RNA samples mixed in equal amounts. Two replicates of each experiment were performed using different microarray slides, in which sample and reference RNAs, labeled either with Cy3 or Cy5 fluorochromes, were crossed in both combinations (dye-swap** procedure). miRNAs were labeled with the mirVana Labeling Kit (Ambion) and amine-reactive dyes (GE Healthcare) as recommended by the manufacturer's protocol [45]. Normalization of expression levels of all spot replicates was performed by MIDAW [50]. Principal component analysis, cluster analysis and profile similarity searching were performed with tMev software [51]. One and two class Significance Analysis of Microarray (SAM) allowed to identify differentially expressed miRNAs [52].
Capture probe and its coupling to microspheres
A sequence of 21-23 nt complementary for each tested miRNAs (listed in the Table 1) was chosen as capture probe and synthesized with 5'-amino linker and a C12 spacer (PRIMM, Milan, Italy). Capture-probe oligonucleotides were covalently linked to carboxylated fluorochrome microspheres (Bio-Rad Laboratories, Hercules, CA, USA) in water-soluble carbodiimide. Specifically, 1 × 106 carboxylated microspheres were pelleted in a microcentrifuge for 5 minutes at 12,000×g and then supernatant was carefully removed. The dry microspheres were dissolved in 20 μl of a buffer containing 0.1 M MES (Sigma-Aldrich, St. Louis, MO, USA) at pH 4.5. The amino-substituted capture probe was dissolved in molecular biology grade water at a concentration of 100 μM and 0.5 μl of the solution (containing 0.05 nmole of capture probe oligonucleotides) was added to the beads for the coupling reaction. The coupling reaction was performed by adding 2.5 μl of a freshly made solution of 10 mg/ml 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)(Pierce, Thermo Scientific, Wilmington, DE, USA) in molecular biology grade water. The mixture of microspheres, capture probes, and EDC was vortexed briefly and incubated at room temperature for 30 minutes in the dark. Occasionally, the reaction was mixed by finger flicking the tube to keep the microspheres in suspension. A second incubation steps was done adding a freshly-made solution of 10 mg/mL EDC in molecular biology grade water. After the coupling reaction, 500 μl of 0.02% Tween 20 (Sigma-Aldrich) was added to the microspheres. The solution was mixed well by vortex and centrifuged for 6 minutes at 12,000×g. The supernatant containing free-capture-probe oligonucleotides and excess EDC was carefully removed. The coupled microspheres were washed in 500 μl of 0.1% SDS (Sigma-Aldrich) by vortex and centrifuged for 5 minutes at 12,000×g. Finally, the supernatant was removed and the capture-probe conjugated microspheres were resuspended in 20 μl of TE pH 8.0 and stored at 4°C in a dark box (stable for at least 6 months). The microspheres were diluted in TE buffer and counted using a Bürker chamber under the microscope at 100× magnification.
miRNA labeling
At the beginning, 1 μg of small RNA molecules (< 200-nt) and 200 pg of a synthetic pre-labeling control RNA (5'-UCUUAGUCUUGAUUGUGGCAAUG-3', PRIMM) were mixed in order to control target preparation efficiency and to normalize expression data. The mixture was polyadenylated using Poly(A) Tailing Kit (Ambion) according to the manufactures' instructions. The reaction was precipitated with NaOAc 3 M pH 5.5 (1/10 volume) and absolute ethanol (4 volumes) overnight at -20°C. The polyadenylated RNA molecules were resuspended in 15 μl of H2O RNase free and then volume was reduced to 3.2 μl by vacuum (VR-1, Heto-Holten, Denmark). miRNAs labeling was performed by mRAP modified protocol in which miRNA-derived cDNAs were flanked by synthesized oligomers at each end. The SMART (switching mechanism at the 5'-end of RNA templates of reverse transcriptase) oligo sequence (SMART-16attB1-T3: 5'-TACAAAAAAGCAGGCTAATTAACCCTCACTAAAggg-3') and the overhang of the oligo-dT15-T7 primer (5'-GTGAATTGTTAATACGACTCACTATAGGCGC [dT]15N-3') were used for first strand synthesis. First strand cDNA synthesis was performed from 500 ng of small RNA in a 10 μl reaction. Then, the reaction was diluted 1:2 and incubated at 72°C for 7 min. Second strand reaction mix was added to 3.0 μl of diluted first strand cDNA to give a final concentration of 1X BD Advantage 2 PCR reaction buffer (Clontech Laboratories, Mountain View, CA, USA), 0.2 mM dNTPs, 100 nM primers (T3-biotinylated forward primer: 5' - biotAATTAACCCTCACTAAAGGG-3' and T7 reverse primer: 5'-TAATACGACTCACTATAGG-3') and 1X of Advantage 2 DNA polymerase mix (Clontech Laboratories) in a total volume of 25 μl. This second strand reaction mixture was incubated for 22 cycles of the following steps: 15 sec 95°C, 20 sec 51°C and 20 sec 72°C. Only those ss cDNAs having a SMART anchor sequence at the 5'-end were used as template and exponentially amplified. The second strand reaction was precipitated in sodium acetate-ethanol solution and dissolved in 11 μl TE buffer pH 8.0 (10 mM TrisHCl pH 8.0, 1 mM EDTA). Biotinylated cDNA quantity was assessed by Nanodrop spectrophotometer (Nanodrop Technologies) and stored at -20°C until hybridization with microspheres. Labelled target produced by a single PCR reaction was sufficient for two hybridization reactions.
Hybridization of targets to capture probes coupled to microspheres
The microspheres of each probe set were resuspended by vortexing for approximately 20 seconds. A microsphere mixture was prepared by diluting coupled stocks to 150 microspheres of each set/μl in 1.5× TMAC Hybridization Buffer (5 M tetramethylammonium chloride, 0.15% Sarkosyl; 75 mM Tris-HCl pH 8.0; and 6 mM EDTA, pH 8.0), followed by vortex mixing for approximately 20 seconds. Two μg of biotinylated DNA target in 17 μL of TE buffer pH 8.0 was added to 33 μl of microsphere mixture (approximately 5,000 beads per color) in the wells of a 96-well plate. 17 μl of TE buffer pH 8.0 were added to background wells. Each well reaction was mixed gently by pipetting up and down several times and the labeled DNA was denatured by heating at 95-100°C for 3 min. The hybridization mixture was incubated at 48°C for 17 h, covering the plate to prevent evaporation, in a Eppendorf microplate incubator with shaking speed of 700 rpm. After incubation, the hybridization mixture was spun down for 3 min at 3,000×g to pellet the microspheres. Supernatant was carefully removed with a pipette without disturbing the microspheres. During centrifugation, fresh reporter mix was prepared by diluting streptavidin-conjugated R-phycoerythrin (Invitrogen) to 3 mg/ml in 1× TMAC Hybridization Buffer and 75 μl of reporter mix were added to the microspheres. The solution was gently mixed by pipetting and incubated in the dark at 48°C for 15 minutes in a Eppendorf microplate incubator. 50 μl of each sample were transferred to a Multiscreen HTS plate (Millipore) and analyzed on the BioPlex™ (Luminex® 100™, BioRad) machine at hybridization temperature.
Bead-based detection
Each set of microspheres was distinguished by assigned colour code (different percentage of red and orange) inside the microspheres. In our experiments we have used four different probe sets (regions: 1, 21, 51 e 57). The fluorescence associated to the surface of each bead, corresponding to the amounts of bound miRNAs, was detected and measured by the laser detector. The Bio-Plex™ (Luminex® 100™, Bio-Rad Laboratories) system detects fluorescent dyes with an excitation wavelength of -532 nm and emission wavelength -580 nm. For each experiment, 100 events of each subset of microspheres were analyzed on the Bio-Plex™ system to obtain a median fluorescence intensity value (MFI) that was representative of the whole population of each set of beads.
Computational analyses (data processing and quality control)
To eliminate bead-specific background, the reading of every bead for every samples was first processed by subtracting the average readings of that particular bead in the absence of target miRNAs. Samples with median fluorescence intensity values smaller than background signals were removed. Every samples was assayed in three wells. Each of the three wells contained 4 probes: miR-23a, miR-27a, miR-199a and one pre-labeling control (Spike-18). Expression data were scaled according to the pre-labeling control in order to normalize readings from different probe/bead sets for the same sample and to normalize for the labeling efficiency. Technical replicate samples for each probe were summarized by their mean profile and expression data (test/control) were log2 transformed. The error associated to each probe is obtained by quadratic propagation from standard deviation.
qRT-PCR TaqMan
TaqMan® MicroRNA Assays incorporate a target-specific stem-loop, reverse transcription primer. The stem-loop structure provides specificity only for the mature miRNA target and forms a RT primer/mature miRNA-chimera that extends the 5'-end of the miRNA. The resulting longer RT amplicon presents a template amenable to standard real-time PCR using TaqMan Assays [22]. In brief, according to the manufacture's instructions (Applied Biosystems), each 15 μl RT reaction contained purified 10 ng of total RNA, 3.0 μl of 5× stem-loop RT primer, 1× RT buffer, 0.25 mM each of dNTPs, 50 U MultiScribe™ reverse transcriptase and 3.8 U RNase inhibitor. The reactions were incubated in a Mastercycler EP gradient S (Eppendorf) in 0.2 ml PCR tubes for 30 min at 16°C, 30 min at 42°C, followed by 5 min at 85°C, and then held at 4°C. RT products were diluted two times with H2O prior to setting up PCR reaction. Each real-time PCR for each miRNA assay (10 μl volume) was carried out in triplicate, and each 10 μl reaction mixture included 1 μl of diluted RT product, 5 μl of 2 × TaqMan® Universal PCR Master Mix and 0.5 μl of 20× TaqMan® MicroRNA Assay. The reaction was incubated in a 7500 Real-Time PCR System (Applied Biosystems) in 96- well plates at 95°C for 10 min, followed by 40 cycles of the following steps: 95°C for 15 sec and 60°C for 1 min. The threshold cycle (CT) is defined as the fractional cycle number at which the fluorescence exceeds the fixed threshold of 0.2. To evaluate differences in miRNA expression, a relative quantification method was chosen where the expression of the miRNA target is standardized by a non-regulated small non-coding RNA used as reference.
Consequently, three replicates of each sample and endogenous control were amplified. U6B small nuclear (RNU6B) was used as endogenous control because the level of this small RNA remains essentially constant from sample to sample. To calculate the relative expression ratio, the 2-Δ ΔCt (RQ, relative quantification) method implemented in the 7500 Real Time PCR System software [53] was used. This method determines the change in expression of a nucleic acid sequence (target) in a test sample relative to the same sequence in a calibrator sample.
References
Engels BM, Hutvagner G: Principles and effects of microRNA-mediated post-transcriptional gene regulation. Oncogene 2006, 25: 6163-6169. 10.1038/sj.onc.1209909 10.1038/sj.onc.1209909
Lippman Z, Martienssen R: The role of RNA interference in heterochromatic silencing. Nature 2004, 431: 364-370. 10.1038/nature02875 10.1038/nature02875
Zhang B, Wang Q, Pan X: MicroRNAs and their regulatory roles in animals and plants. J Cell Physiol 2007, 210: 279-289. 10.1002/jcp.20869 10.1002/jcp.20869
Harfe BD: MicroRNAs in vertebrate development. Curr Opin Genet Dev 2005, 15: 410-415. 10.1016/j.gde.2005.06.012 10.1016/j.gde.2005.06.012
** Y, Shalgi R, Fodstad O, Pilpel Y, Ju J: Differentially regulated micro-RNAs and actively translated messenger RNA transcripts by tumor suppressor p53 in colon cancer. Clin Cancer Res 2006, 12: 2014-2024. 10.1158/1078-0432.CCR-05-1853 10.1158/1078-0432.CCR-05-1853
Bartel DP: MicroRNAs: genomics biogenesis, mechanism, and function. Cell 2004, 116: 281-297. 10.1016/S0092-8674(04)00045-5 10.1016/S0092-8674(04)00045-5
Stefani G, Slack FJ: Small non-coding RNAs in animal development. Nat Rev Mol Cell Biol 2008, 9: 219-230. 10.1038/nrm2347 10.1038/nrm2347
Lee RC, Ambros V: An extensive class of small RNAs in Caenorhabditis elegans. Science 2001, 294: 862-864. 10.1126/science.1065329 10.1126/science.1065329
Jaubert S, Mereau A, Antoniewski C, Tagu D: MicroRNAs in Drosophila: the magic wand to enter the Chamber of Secrets? Biochimie 2007, 89: 1211-1220. 10.1016/j.biochi.2007.05.012 10.1016/j.biochi.2007.05.012
Landgraf P, Rusu M, Sheridan R, Sewer A, Iovino N, Aravin A, Pfeffer S, Rice A, Kamphorst AO, Landthaler M, Lin C, Socci ND, Hermida L, Fulci V, Chiaretti S, Foà R, Schliwka J, Fuchs U, Novosel A, Müller RU, Schermer B, Bissels U, Inman J, Phan Q, Chien M, Weir DB, Choksi R, De Vita G, Frezzetti D, Trompeter HI, et al.: A mammalian microRNA expression atlas based on small RNA library sequencing. Cell 2007, 129: 1401-1414. 10.1016/j.cell.2007.04.040 10.1016/j.cell.2007.04.040
Pasquinelli AE, Ruvkun G: Control of developmental timing by microRNAs and their targets. Annu Rev Cell Dev Biol 2002, 18: 495-513. 10.1146/annurev.cellbio.18.012502.105832 10.1146/annurev.cellbio.18.012502.105832
Rana TM: I lluminating the silence: understanding the structure and function of small RNAs. Nat Rev Mol Cell Biol 2007, 8: 23-36. 10.1038/nrm2085 10.1038/nrm2085
Bentwich I, Avniel A, Karov Y, Aharonov R, Gilad S, Barad O, Barzilai A, Einat P, Einav U, Meiri E, Sharon E, Spector Y, Bentwich Z: Identification of hundreds of conserved and nonconserved human microRNAs. Nat Genet 2005, 37: 766-770. 10.1038/ng1590 10.1038/ng1590
Lewis BP, Shih IH, Jones-Rhoades MW, Bartel DP, Burge CB: Prediction of mammalian microRNA targets. Cell 2003, 115: 787-798. 10.1016/S0092-8674(03)01018-3 10.1016/S0092-8674(03)01018-3
Wang X, El Naqa IM: Prediction of both conserved and nonconserved microRNA targets in animals. Bioinformatics 2008, 24: 325-332. 10.1093/bioinformatics/btm595 10.1093/bioinformatics/btm595
Reinhart BJ, Slack FJ, Basson M, Pasquinelli AE, Bettinger JC, Rougvie AE, Horvitz HR, Ruvkun G: The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans . Nature 2000, 403: 901-906. 10.1038/35002607 10.1038/35002607
Valoczi A, Hornyik C, Varga N, Burgyan J, Kauppinen S, Havelda Z: Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes. Nucleic Acids Res 2004, 32: e175. 10.1093/nar/gnh171 10.1093/nar/gnh171
Berezikov E, Cuppen E, Plasterk RH: Approaches to microRNA discovery. Nat Genet 2006,38(Suppl):S2-7. 10.1038/ng1794 10.1038/ng1794
Takada S, Berezikov E, Yamashita Y, Lagos-Quintana M, Kloosterman WP, Enomoto M, Hatanaka H, Fujiwara S, Watanabe H, Soda M, Choi YL, Plasterk RH, Cuppen E, Mano H: Mouse microRNA profiles determined with a new and sensitive cloning method. Nucleic Acids Res 2006, 34: e115. 10.1093/nar/gkl653 10.1093/nar/gkl653
Mineno J, Okamoto S, Ando T, Sato M, Chono H, Izu H, Takayama M, Asada K, Mirochnitchenko O, Inouye M, Kato I: The expression profile of microRNAs in mouse embryos. Nucleic Acids Res 2006, 34: 1765-1771. 10.1093/nar/gkl096 10.1093/nar/gkl096
Schmittgen TD, Jiang J, Liu Q, Yang L: A high-throughput method to monitor the expression of microRNA precursors. Nucleic Acids Res 2004, 32: e43. 10.1093/nar/gnh040 10.1093/nar/gnh040
Chen C, Ridzon DA, Broomer AJ, Zhou Z, Lee DH, Nguyen JT, Barbisin M, Xu NL, Mahuvakar VR, Andersen MR, Lao KQ, Livak KJ, Guegler KJ: Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 2005, 33: e179. 10.1093/nar/gni178 10.1093/nar/gni178
Cummins JM, He Y, Leary RJ, Pagliarini R, Diaz LA Jr, Sjoblom T, Barad O, Bentwich Z, Szafranska AE, Labourier E, Raymond CK, Roberts BS, Juhl H, Kinzler KW, Vogelstein B, Velculescu VE: The colorectal microRNAome. Proc Natl Acad Sci USA 2006, 103: 3687-3692. 10.1073/pnas.0511155103 10.1073/pnas.0511155103
Lu J, Getz G, Miska EA, Alvarez-Saavedra E, Lamb J, Peck D, Sweet-Cordero A, Ebert BL, Mak RH, Ferrando AA, Downing JR, Jacks T, Horvitz HR, Golub TR: MicroRNA expression profiles classify human cancers. Nature 2005, 435: 834-838. 10.1038/nature03702 10.1038/nature03702
Jay C, Nemunaitis J, Chen P, Fulgham P, Tong AW: miRNA profiling for diagnosis and prognosis of human cancer. DNA Cell Biol 2007, 26: 293-300. 10.1089/dna.2006.0554 10.1089/dna.2006.0554
Yin JQ, Zhao RC, Morris KV: Profiling microRNA expression with microarrays. Trends Biotechnol 2008, 26: 70-76. 10.1016/j.tibtech.2007.11.007 10.1016/j.tibtech.2007.11.007
Rosa A, Brivanlou AH: MicroRNAs in early vertebrate development. Cell Cycle 2009, in press.
Kloosterman WP, Plasterk RH: The diverse functions of microRNAs in animal development and disease. Dev Cell 2006, 11: 441-450. 10.1016/j.devcel.2006.09.009 10.1016/j.devcel.2006.09.009
Asli NS, Pitulescu ME, Kessel M: MicroRNAs in organogenesis and disease. Curr Mol Med 2008, 8: 698-710. 10.2174/156652408786733739 10.2174/156652408786733739
Esquela-Kerscher A, Slack FJ: Oncomirs: microRNAs with a role in cancer. Nature Rev Cancer 2006, 6: 259-269. 10.1038/nrc1840 10.1038/nrc1840
Croce CM: Causes and consequences of microRNA dysregulation in cancer. Nat Rev Genet 2009, 10: 704-714. 10.1038/nrg2634 10.1038/nrg2634
Drakaki A, Iliopoulos D: MicroRNA Gene Networks in Oncogenesis. Curr Genomics 2009, 10: 35-41. 10.2174/138920209787581299 10.2174/138920209787581299
Latronico MV, Condorelli G: MicroRNAs and cardiac pathology. Nat Rev Cardiol 2009, 6: 419-29. 10.1038/nrcardio.2009.56 10.1038/nrcardio.2009.56
Pauley KM, Chan EK: MicroRNAs and their emerging roles in immunology. Ann N Y Acad Sci 2008, 1143: 226-239. 10.1196/annals.1443.009 10.1196/annals.1443.009
Bentwich I, Avniel A, Karov Y, Aharonov R, Gilad S, Barad O, Barzilai A, Einat P, Einav U, Meiri E, Sharon E, Spector Y, Bentwich Z: Identification of hundreds of conserved and nonconserved human microRNAs. Nat Genet 2005, 37: 766-770. 10.1038/ng1590 10.1038/ng1590
Li SC, Pan CY, Lin WC: Bioinformatic discovery of microRNA precursors from human ESTs and introns. BMC Genomics 2006, 7: 164. 10.1186/1471-2164-7-164 10.1186/1471-2164-7-164
Earley MC, Vogt RF Jr, Shapiro HM, Mandy FF, Kellar KL, Bellisario R, Pass KA, Marti GE, Stewart CC, Hannon WH: Report from a workshop on multianalyte microsphere assays. Cytometry 2002, 50: 239-42. 10.1002/cyto.10140 10.1002/cyto.10140
Luminex xMAP Technology[http://www.luminexcorp.com/technology/index.html]
Blenkiron C, Goldstein LD, Thorne NP, Spiteri I, Chin SF, Dunning MJ, Barbosa-Morais NL, Teschendorff AE, Green AR, Ellis IO, Tavaré S, Caldas C, Miska EA: MicroRNA expression profiling of human breast cancer identifies new markers of tumor subtype. Genome Biol 2007, 8: R214. 10.1186/gb-2007-8-10-r214 10.1186/gb-2007-8-10-r214
Taylor JD, Briley D, Nguyen Q, Long K, Iannone MA, Li MS, Ye F, Afshari A, Lai E, Wagner M, Chen J, Weiner MP: Flow cytometric platform for high-throughput single nucleotide polymorphism analysis. BioTechniques 2001, 30: 661-666. 668-669
Prabhakar U, Eirikis E, Davis HM: Simultaneous quantification of proinflammatory cytokines in human plasma using the LabMAP assay. Journal of immunological methods 2002, 260: 207-218. 10.1016/S0022-1759(01)00543-9 10.1016/S0022-1759(01)00543-9
Mano H, Takada S: mRAP, a sensitive method for determination of microRNA expression profiles. Methods 2007, 43: 118-122. 10.1016/j.ymeth.2007.04.006 10.1016/j.ymeth.2007.04.006
Takada S, Mano H: Profiling of microRNA expression by mRAP. Nature protocols 2007, 2: 3136-3145. 10.1038/nprot.2007.457 10.1038/nprot.2007.457
Merlino G, Helman LJ: Rhabdomyosarcoma-working out the pathways. Oncogene 1999, 18: 5340-348. 10.1038/sj.onc.1203038 10.1038/sj.onc.1203038
Shingara J, Keiger K, Shelton J, Laosinchai-Wolf W, Powers P, Conrad R, Brown D, Labourier E: An optimized isolation and labeling platform for accurate microRNA expression profiling. RNA 2005, 11: 1461-1470. 10.1261/rna.2610405 10.1261/rna.2610405
Anderson J, Gordon T, McManus A, Mapp T, Gould S, Kelsey A, McDowell H, Pinkerton R, Shipley J, Pritchard-Jones K: Detection of the PAX3-FKHR fusion gene in paedriatic Rhabdomyosarcoma: a reproducible predictor of outcome? Br J Cancer 2001, 85: 831-35. 10.1054/bjoc.2001.2008 10.1054/bjoc.2001.2008
De Pittà C, Tombolan L, Albiero G, Sartori F, Romualdi C, Jurman G, Carli M, Furlanello C, Lanfranchi G, Rosolen A: Gene expression profiling identifies potential relevant genes in alveolar rhabdomyosarcoma pathogenesis and discriminates PAX3-FKHR positive and negative tumors. Int J Cancer 2006, 118: 2772-81. 10.1002/ijc.21698 10.1002/ijc.21698
Romualdi C, De Pittà C, Tombolan L, Bortoluzzi S, Sartori F, Rosolen A, Lanfranchi G: Defining the gene expression signature of rhabdomyosarcoma by meta-analysis. BMC Genomics 2006, 7: 287. 10.1186/1471-2164-7-287 10.1186/1471-2164-7-287
Davicioni E, Finckenstein FG, Shahbazian V, Buckley JD, Triche TJ, Anderson MJ: Identification of a PAX-FKHR gene expression signature that defines molecular classes and determines the prognosis of alveolar rhabdomyosarcomas. Cancer Res 2006, 66: 6936-46. 10.1158/0008-5472.CAN-05-4578 10.1158/0008-5472.CAN-05-4578
Romualdi C, Vitulo N, Del Favero M, Lanfranchi G: MIDAW: a web tool for statistical analysis of microarray data. Nucleic Acids Res 2005, (33 Web Server):W644-9. 10.1093/nar/gki497
Saeed AI, Bhagabati NK, Braisted JC, Liang W, Sharov V, Howe EA, Li J, Thiagarajan M, White JA, Quackenbush J: TM4 microarray software suite. Methods in Enzymology 2006, 411: 134-93. 10.1016/S0076-6879(06)11009-5 10.1016/S0076-6879(06)11009-5
Tusher VG, Tibshirani R, Chu G: Diagnosis of multiple cancer types by shrunken centroids of gene expression. Proc Natl Acad Sci USA 2001, 98: 5116-121. 10.1073/pnas.091062498 10.1073/pnas.091062498
Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001, 25: 402-408. 10.1006/meth.2001.1262 10.1006/meth.2001.1262
Acknowledgements
This work was supported by Associazione Italiana per la Ricerca sul Cancro (AIRC) and Biotech Action III bis (CIPE 3/06 DGR 4073 19/12/2006 Veneto Region). The authors wish to thank Emanuele Papini, Paola Cecchini and Regina Tavano (Dept. Biomedical Sciences and C.R.I.B.I. Biotechnology Centre-University of Padova, Italy) for technical support in bead-based detection with BioPlex. We are also grateful to Angelica Zin for RMS cell lines.
Author information
Authors and Affiliations
Corresponding authors
Additional information
Authors' contributions
AB performed small RNA molecules isolation, capture probes coupling to microspheres, miRNA labeling method, bead-based detection with BioPlex and computational analyses (data processing and quality control). SC performed RMS cell lines culture, small RNA molecules isolation, microarray experiments and qRT-PCR validation. SC participated in conceiving the study and in development of miRNA labelling method. LT performed RMS cell lines culture and participated in total RNA extraction. AR provided RMS cell lines and revised the manuscript. GL supervised the study, participating in the design and coordination of the work, the interpretation of the results and revision of the manuscript. CDP conceived and supervised the study, participating in the design and coordination of the work, the interpretation of data and manuscript writing. All Authors read and approved the final version of the manuscript declaring that they have no potential conflicts of interests.
Authors’ original submitted files for images
Below are the links to the authors’ original submitted files for images.
Rights and permissions
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
About this article
Cite this article
Biscontin, A., Casara, S., Cagnin, S. et al. New miRNA labeling method for bead-based quantification. BMC Molecular Biol 11, 44 (2010). https://doi.org/10.1186/1471-2199-11-44
Received:
Accepted:
Published:
DOI: https://doi.org/10.1186/1471-2199-11-44