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Blood, Vol. 111, Issue 6, 3183-3189, March 15, 2008
MicroRNA signatures associated with cytogenetics and prognosis in acute myeloid leukemia
Blood Garzon et al.
111: 3183
Supplemental materials for Garzon et al
Supplemental materials and methods MicroRNA (miRNA) microarrays
Five micrograms of total RNA was used for hybridization on the miRNA microarray chips in quadruplicate with probes corresponding to the 250 human mature and precursor miRNAs (as described in the miRBase (http://microrna.sanger.ac.uk) in November 2005).1 The total RNA was separately added to reaction mix in a final volume of 12 µl, containing 1 µg of 3′-(N)8-(A)12-biotin-(A)12-biotin-5′ random oligonucleotide primer. The mixture was incubated for 10 min at 70°C and chilled on ice. With the mixture remaining on ice, 4 µl of 5× first-strand buffer, 2 µl of 0.1 M DTT, 1 µl of 10 mM dNTP mix, and 1 µl of SuperScript II RNaseH− reverse transcriptase (200 units/µl) were added to a final volume of 20 µl, and the mixture was incubated for 90 min in a 37°C water bath. After incubation for first-strand cDNA synthesis, 3.5 µl of 0.5 M NaOH/50 mM EDTA was added into 20 µl of first-strand reaction mix and incubated at 65°C for 15 min to denature the RNA/DNA hybrids and degrade RNA templates. Then, 5 µl of 1 M Tris·HCI (pH 7.6, Sigma) was added to neutralize the reaction mix, and labeled targets were stored at −80°C prior to hybridization. The microarrays were hybridized in 6× SSPE (0.9 M sodium chloride/60 mM sodium phosphate/8 mM EDTA, pH 7.4)/30% formamide at 25°C for 18 h, washed in 0.75× TNT (Tris·HCl/sodium chloride/Tween 20) at 37°C for 40 min, and processed by using direct detection of the biotin-containing transcripts by Streptavidin-Alexa647 conjugate. Processed slides were scanned using a GenePix Axon 4000B microarray scanner, with the laser set to 635 nm, at fixed PMT setting of 800, and a scan resolution of 10 mm. In addition to the miRNA probes, oligonucleotides for eight human TRNAs and 3 snRNAs by using similar design criteria were included. (Table S1). Data analysis
After obtaining the slides images using GenePix Pro, average values of the replicate spots of each miRNA were background-subtracted, normalized and subject to further analysis. Spots flagged as absent or outliers according to the GenePix Pro quality control were not included in the analysis. BRB Array Tools was used for normalization. As single-channel experiments, the arrays were normalized to a reference array, so that the difference in log-intensities between the array and reference array had median of zero over the set of housekeeping genes. The reference array was automatically chosen as the median array (the array whose median log-intensity value is the median over all median log-intensity values for the complete set of arrays). The housekeeping genes normalization was performed by computing the gene-by-gene difference between each array and the reference array, and subtracting the median difference over housekeeping genes from the log-intensities on that array. The “housekeeping” non coding genes were selected because they are non-coding as the miRNA genes (table S1). We extended the version 1 tRNA genes to include U2, U4, U6 small non-coding RNA genes and GAPDH mRNA. U6 are extensively used in miRNA papers from different labs for normalization of Northern blots. Due to the heterogeneity of AML, the miRNAs were retained when present in at least 20% of samples. Absent calls were thresholded to 22 (4.5 in log2 scale) prior to statistical analysis. This level is the average minimum intensity level detected above background in miRNA chips experiments. MiRNA nomenclature was according to the miRNA database at Sanger Center1. Differentially expressed miRNAs were identified by using the adjusted t test procedure within significance analysis of microarrays (SAM).2 The SAM 2.0 application with a threshold difference in expression set to 2, s0 percentile set to 0.05 (default) and the number of permutations set to 100 (default). The SAM Excel plug-in used here calculates a score for each gene on the basis of the change in expression relative to the standard deviation of all measurements. Since this is a multiple test, permutations are performed to calculate the false discovery rate (FDR) or q-value. MiRNAs with FDRs less than 5% and fold changes more than 2 were considered for further analysis. The microarray dataset is deposited in Array-Express (http://www.ebi.ac.uk/arrayexpress). MiRNA qRT-PCR validation
The single tube TaqMan miRNA Assays was used to detect and quantify mature miRNAs on Applied Biosystems Real-Time PCR instruments in accordance with manufacturer's instructions (Applied Biosystems, Foster City, CA). Normalization was performed with the invariant let-7i (Applied Biosystems). All RT reactions, including no-template controls and RT minus controls, were run in a GeneAmp PCR 9700 Thermocycler (Applied Biosystems).Gene expression levels were quantified using the ABI Prism 7900HT Sequence detection system (Applied Biosystems). Comparative real-time PCR was performed in triplicate, including no-template controls. Relative expression was calculated using the comparative Ct method.3 To validate the microarray data we used Pearson correlation and linear regression analysis (SPSS package) using 42 miRNA measurements in 12 patients. These functions examine each pair of measurements (one from the chip and the other from qRT-PCR) to determine whether the two variables tend to move together, that is whether the larger values from the chip (high expression) are associated with the higher values from the qRT-PCR (2ΔCt).
Files in this Data Supplement:
- Table S6. MicroRNAs differentially expressed in normal karyotype AML patients compared with abnormal karyotype AML (PDF, 19.6 KB) -
All miRNAs, except miR-368, miR-191 and miR-192 were found also differentially expressed in treated AML patients with normal karyotype (10) compared with treated AML patients with abnormal karyotype (38). MiRNAs in red are up-regulated, in green down-regulated.
- Table S7. Clinical characteristics of 54 treated AML patient samples (relapsed n= 34 or primary refractory n= 20) (PDF, 68.9 KB)
- Table S8. MicroRNAs differentially expressed in treated patients with t(11q23) compared with other treated AML patients with other cytogenetic abnormalities including normal karyotype (PDF, 28.2 KB)
- Table S9. MicroRNAs differentially expressed in normal karyotype treated AML patients compared with abnormal karyotype treated AML patients (PDF, 29.3 KB)
- Table S10. MicroRNAs up-regulated in treated AML patients with FLT3-ITD mutations vs. FLT3-wt (PDF, 13.7 KB)
- Figure S1. Validation of microarray data by qRT-PCR (JPG, 33.8 KB)
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Scatter plot showing the positive correlation between the miRNA microarrays expression values and the normalized qRT-PCR after 2ΔCt conversion for each sample. The solid pink line represents the predicted Y, while the blue dots are patient samples. The lower the qRT-PCR (ΔCt values), the lower the expression level of the miRNA.
- Figure S2. Validation of the microarray results for selected miRNAs in patients with t(9;11) (JPG, 28.3 KB)
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Average miR-326 (A) and miR-29a, miR-29b and miR-29c (B) expression in newly diagnosed AML patients with t(9;11) (n=3) and non 11q23 AML (n=10) measured by qRT-PCR. The miRNA expression between the different groups was compared using t-test (SPSS).
- Figure S3. Validation of the microarray results for selected miRNAs in patients with normal karyotype (JPG, 31.1 KB)
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Average miR-10a (A), miR-126 (B) and miR-30c (C) expression in newly diagnosed AML patients with normal karyotype (n=12) and abnormal karyotype (n=22) measured by qRT-PCR. The miRNA expression between the different groups was compared using t-test (SPSS).
REFERENCES
1. Griffiths-Jones S. The microRNA registry. Nucleic Acids Res. 2004; 32, Database issue D109-D111.
2. Tusher VG, Tibshirani R, Chu G. Significant analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci USA. 2001; 98:5116:21.
3. 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.
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