SUPPLEMENTARY MATERIALS AND METHODS
Allele-specific regulation of DISC1 expression by miR-135b-5p
Mari Rossi, Helena Kilpinen, Mikko Muona, Ida Surakka, Catherine Ingle, Jenni Lahtinen, William Hennah,
Samuli Ripatti, Iiris Hovatta
Contents
Polymorphic miRNA target prediction .................................................................................................................1
Cell culture............................................................................................................................................................1
Reporter gene constructs .....................................................................................................................................1
Transfections ........................................................................................................................................................2
RNA extraction......................................................................................................................................................3
cDNA synthesis .....................................................................................................................................................3
Quantitative Real-Time PCR (qRT-PCR) ................................................................................................................4
Luciferase assays ..................................................................................................................................................5
Quality control of the data ...................................................................................................................................5
Statistical analysis .................................................................................................................................................6
References ............................................................................................................................................................7
Polymorphic miRNA target prediction
Two databases, Patrocles (http://www.patrocles.org)1 and PolymiRTS (http://compbio.uthsc.edu/miRSNP/)2
were used to search for polymorphic miRNA target sites in the human DISC1 gene. At the time of this study,
only four DISC1 isoforms were properly annotated (L="long" [NM_018662.2], Lv="long variant"
[NM_001012957.1], S="short" [NM_001012959.1], and Es="extra short" [NM_001012958.1]). Thus, the target
predictions include only these transcripts, and for the purpose of this study, we further limited our search to
the two full-length isoforms, L and Lv.
Cell culture
HEK293FT cells (Invitrogen Inc, Carlsbad, CA, USA) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Carlsbad, CA, USA) with L-glutamine, 4500 mg/L D-Glucose, and 110 mg/L sodium pyruvate. The
medium was supplied with 10% FBS, 4mM L-glutamine or glutaMAX, 1% penicillin, and 1% streptomycin at
+37°C with 5% CO2.
Reporter gene constructs
The complete 3’UTR sequence (4441 bp) of DISC1 Lv isoform (IMAGE:900180)3 was cloned into a pmirGLO dualluciferase reporter vector (Promega, Madison, WI, USA) by creating a suitable artificial SacII restriction site
using short oligoduplexes (all oligonucleotide and primer sequences available upon request from IH). In
addition, we created four reporter constructs harbouring only the desired miRNA binding sites and
approximately 60 nt of flanking sequences with either allele for SNPs rs11122396 (A/G) or rs11803088 (C/T).
The binding site oligoduplexes were generated according to the manufacturer’s instructions (Promega) and
cloned into the XbaI/SacI site of the vector. Full-length DISC1 3’UTR construct harbouring the alternative allele
(G) for rs11122396 was created using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent
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Technologies, Santa Clara, CA, USA) according to the manufacturer’s instructions. Mutagenesis primers were
designed using QuikChange Primer Design Program (http://www.stratagene.com/qcprimerdesign). Orientation
of the correct inserts and presence of the alternative alleles was confirmed by restriction analysis and/or direct
sequencing of the constructs.
Transfections
a) Transfections for qRT-PCR of endogenous DISC1
HEK293FT cells were split into antibiotic-free D-MEM medium 48 h prior to transfections. The transient
transfections were performed in 96-well format using 0.5 μl of siPORT NeoFX reverse transfection reagent
(Ambion Inc, Austin, TX, USA) and 6000 cells per well in a total volume of 100 μl. Each transfection experiment
contained untransfected cells and cells transfected with either selected pre-miRNA precursor molecules
(Ambion Inc.), an siRNA against DISC1 (Sigma MISSION® siRNA-A ID: SASI_HS01_00239305, Sigma-Aldrich, St.
Louis, MO, USA), or a negative control miRNA (control #1; Ambion Inc, ID: 17110) to monitor for unspecific
technical artefacts. The final concentration of small RNAs per reaction was 30 nM. Plain D-MEM (Gibco) was
used as the diluent for all reagents. The transfection complexes (20 μl) were prepared according to the
manufacturer’s instructions, distributed to the plate, overlaid with the cells, and assayed after 48 h. Each
experiment contained three biological replicates in triplicate for each tested small-RNA. Each experiment was
carried out three independent times.
b) Transfections for reporter gene experiments
HEK293FT cells were split into antibiotic-free D-MEM medium 24 h prior to transfections. The cells were
transiently co-transfected using either NeoFX transfection reagent (Ambion Inc.) with 30 ng of each reporter
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plasmid, 30 µM of miRNA molecules (miRNA precursors, negative control miRNAs #1 (ID: 17110) and #2 (ID:
17111), or anti-miR miRNA inhibitors (Ambion Inc.)), or 5 nM Silencer® Select siRNA against DISC1 3’UTR
(Invitrogen Inc.), or using the Neon™ Transfection System (Invitrogen Inc.) with 30-100 ng of each reporter
plasmid, 50-100 nM of miRNA molecules, or 50-100 nM of siRNA. Each experiment contained one biological
replicate in triplicate for each tested small-RNA and 20 000 cells per well for chemical transfections or 50 000
cells per well for electroporations. Each experiment was independently carried out at least twice.
RNA extraction
Total RNA from transfected cells was extracted using the TRI Reagent protocol (Molecular Research Center,
Inc., Cincinnati, OH, USA). Cells were lysed with TRI Reagent and the RNA extracted according to the
manufacturer’s instructions. Due to the small amount of starting material, the protocol was supplemented with
manual Phase Lock Gel tubes (5Prime) to aid phase separation. Quality and concentration of the RNA samples
was measured with a Nanodrop spectrophotometer.
cDNA synthesis
Total RNA was treated with DNaseI enzyme (Fermentas, Waltham, MA, USA) prior to cDNA synthesis. 100-500
ng of RNA from each biological replicate was then converted to cDNA using High Capacity RNA-to-cDNA Master
Mix (Applied Biosystems, Carlsbad, CA, USA).
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Quantitative Real-Time PCR (qRT-PCR)
a) qRT-PCR for endogenous DISC1
DISC1 qRT-PCR primers were designed with Primer Express 2.0 (Applied Biosystems) to the boundary of exons 7
and 8 of DISC1. qRT-PCR was performed in 10 µl reaction volume on 384-well Clear Optical reaction plates
(Applied Biosystems) with a 7900HT instrument (Applied Biosystems) using SYBR Green PCR Master Mix
(Applied Biosystems) and the absolute quantification method. Each reaction contained a total of 10 ng of cDNA
template, 5 µl of SYBR-mix, and 0.7 pmol of each primer, and was performed in triplicate on every plate.
Absolute expression values for each sample were extracted based on a five-point standard curve (0.2, 2, 10, 20,
and 50 ng of cDNA) for each primer pair. Human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was
used as the reference gene. Raw data was analyzed using the Sequence Detection Systems (SDS) 2.3 software
(Applied Biosystems). By default, one experiment consisted of three biological replicates analyzed in a single
qRT-PCR run and was repeated three independent times. The thermal profile of each run was as follows: initial
stage of 50°C for 2 min and 95°C for 10 min, 40 cycles of 95°C for 15 s and 60°C for 1 min, followed by a final
dissociation stage of 95°C for 15 s and 60°C for 15 s.
b) TaqMan qRT-PCR for endogenous microRNAs
Endogenous hsa-miR-135b (TaqMan assay ID: 002261) and hsa-miR-559 (ID: 001527) expression levels of
HEK293FT cells relative to human small nucleolar RNA, C/D box 38B (RNU38B) (ID: 001004) were measured
using TaqMan microRNA qRT-PCR assay (Applied Biosystems) according to the manufacturer’s instructions.
Amplification reaction volume was 10 µl containing 1 ng of cDNA, 0,5 µl primers and 10 µl Master mix using
TaqMan Universal PCR Master Mix, no AmpErase UNG reagent (Applied Biosystems). Cycling conditions were
the same as described above. Each sample was amplified in triplicate and the miRNA expression levels were
determined by the comparative threshold cycle (Ct) method and normalized for RNU38B.
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Luciferase assays
Cells were assayed for luciferase activity after 48 h (chemical transfection) or 24 h (electroporation) using DualGlo luciferase assay system (Promega) and PheraStar microplate reader (BMG LABTECH, Offenburg, Germany).
Each 96-well plate represented one biological replicate, and was repeated minimum two times.
Quality control of the data
Careful quality control of the raw data was carried out to ensure high data quality and avoid sources of
experimental bias.
a) Endogenous DISC1 qRT-PCR experiments
The Ct values of all three technical replicates per plate were required to fall within one Ct value, and the
standard deviation (SD) of the Ct values to be < 0.5. PCR efficiency was estimated from the slope of the
standard curve, and the correlation coefficient of the curve (R2) monitored for overall standard quality. A
melting curve analysis was performed at the end of each run to verify specific PCR amplification for the used
primer pair. Negative control wells had no detectable signal.
b) Luciferase assay experiments
SD ≤ 1.0 of the technical replicates was required for all the samples included in the analyses.
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Statistical analysis
a) qRT-PCR experiments for endogenous DISC1
Relative expression values of DISC1 (ratio of DISC1/GAPDH absolute expression values) were
obtained for all three technical replicates separately. A mean of the technical replicates was used to
represent each of the nine biological replicates in the subsequent statistical analyses. The effect of each
miRNA and siRNA was compared to that of the negative control miRNA in the same run. The
statistical significance of the miRNA-induced expression changes was assessed using batch-adjusted
linear regression analysis, with DISC1 relative expression level as the dependent variable. Given the
initial small number of cells for each tested sample, we wanted to be sure that random differences in
RNA extraction efficiency or RNA yield were not influencing the obtained results. Thus, we initially
adjusted the linear model with RNA concentration as well, but it did not have an impact on the results
(data not shown).
b) Luciferase assay experiments
Firefly luciferase activity was normalized to that of the Renilla luciferase for each sample separately. Data for
each reporter gene experiment were averaged from minimum of two independent biological replicates
performed in triplicate. Statistical significance was assessed using the same batch-adjusted linear regression
analysis as for the qRT-PCR data, comparing each miRNA to the average of two negative control miRNAs from
the same run, for each construct separately. Initially we adjusted the linear model also with the transfection
method, but it did not affect the results (data not shown). All statistical analyses were performed using
packages implemented in R2.15.2 (http://www.r-project.org).
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References
1. Hiard S, Charlier C, Coppieters W, Georges M, Baurain D. Nucleic Acids Res. 2010; 38: D640–D651.
2. Bao L, Zhou M, Wu L, Lu L, Goldowitz D, Williams RW et al. Nucleic Acids Res. 2007; 35: D51-4.
3. Lennon G, Auffray C, Polymeropoulos M, Soares MB. Genomics 1996; 33: 151-2.
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