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-swapping procedure). miRNAs were labeled with the mirVana Labeling Kit (Ambion) and amine-reactive dyes (GE Healthcare) as recommended by the manufacturer's protocol . Normalization of expression levels of all spot replicates was performed by MIDAW . Principal component analysis, cluster analysis and profile similarity searching were performed with tMev software . One and two class Significance Analysis of Microarray (SAM) allowed to identify differentially expressed miRNAs .
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.
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.
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.
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 . 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  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.