OCI-AML3 cells were treated with 100 ng/mL SDF-1α or 250 nM POL6326 (a CXCR4 antagonist, Allschwil, Switzerland) up to 8hour. total RNA was extracted at specific time points (0,1, 2, 4, and 8 h), The 0 time point are untreated cells and are used as controls.
Growth protocol
The human AML cell lines OCI-AML3 was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. Cell lines were harvested during the log-phase of growth and seeded at a density of 0.2×106 cells/mL.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted with miRNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol.
Label
biotin
Label protocol
Five micrograms of total RNA were 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′] 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) was 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 in 28.5 μl at -80°C until chip hybridization. Five micrograms of total RNA were 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′] 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) was 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 in 28.5 μl at -80°C until chip hybridization.
Hybridization protocol
Labeled targets from 5 μg of total RNA was used for hybridization on each OSU-CCC miRNA microarray custom version 4, containing 393 mouse, 409 humans and 962 ultraconserved sequences in quadruplicate. All probes on these microarrays are 40-mer oligonucleotides spotted by contacting technologies and covalently attached to a polymeric matrix. 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
Scan protocol
Processed slides were scanned by using a PerkinElmer ScanArray XL5K Scanner with the laser set to 635 nm, at power 80 and PMT 70 settings, and a scan resolution of 10 μm.
Data processing
miRNA microarray images were analyzed using GENEPIX PRO. Average values of the replicate spots of each miRNA were background subtracted; log2-transformed and normalized using quantiles.
