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Supplemental Digital Content 1. Methods, primer sets, and normalization genes for RT-PCR.
Methods
Total RNA was isolated using the miRNeasy Mini Kit (Qiagen, Inc.; Valencia, CA, USA) and
used to generate cDNA templates by reverse transcription of 1 µg RNA using the High Capacity
cDNA Reverse Transcription Kit (Applied Biosystems; Carlsbad, CA, USA) according to the
manufacturer's instructions. The resulting cDNA was diluted 5-fold and 5 µL was used for input
into PCR reactions performed in a total volume of 20 µL containing SensiMix SYBR qPCR
reagents (Bioline USA, Inc.; Tauton, MA, USA) using a Corbett Rotor-Gene 6000 real-time
PCR system (Qiagen, Inc.). For sequences of the primer sets see Table, Supplemental Digital
Content 1. The PCR primers used to detect MOR-1K were designed to target the unique exon 13
in the OPRM1 gene in combination with exon 2 [1]. The pool of C-terminal MOR splice
variants (MOR-1(exons 1-2)) was targeted using a primer set spanning exons 1 and 2 [1], which will
detect most of the MOR variants as they differ primarily in exon 4. For detection of MOR-1(exons
1-2)
and MOR-1K mRNAs, PCR conditions consisted of an initial hold step at 95°C for 10 min
followed by 40 amplification cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 20 s. For
detection of CCL2, CCL8, CCL5, CXCR4, CCR5, and CD4 mRNAs, PCR conditions consisted
of an initial hold step at 95°C for 10 min followed by 40 amplification cycles of 95°C for 5 s,
55°C for 10 s, and 72°C for 20 s. For detection of FLNA mRNA, PCR conditions consisted of
an initial hold step at 95°C for 10 min followed by 40 amplification cycles of 95°C for 10 s,
58°C for 30 s, and 72°C for 30 s. The specificity of the amplified products was verified by
melting curve analysis and agarose gel electrophoresis. Expression levels were normalized to
GAPDH mRNA for untreated individual cell types. For brain tissue from the different subject
2
groups, expression levels were normalized to TATA-binding protein (TBP) mRNA following
assessments of stability comparing various housekeeping genes which included a geNorm
analysis to confirm that TBP was one of the most stably expressed genes in the sample set (see
Figure, Supplemental Digital Content 1). Relative expression was calculated using the 2−ΔΔCt
method [2].
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Primer sets
Gene
Forward primer
Reverse primer
MOR-1(exons 1-2)
MOR-1K
CCL2
CCL8
CCL5
CXCR4
CCR5
CD4
FLNA
GAPDH
TBP
HPRT1
ACTB
EEF2
EIF2B2
SDHA
HMBS
5′- CTTCCTGGTCATGTATGTGATTGTC -3′
5′- GCCAGAGCAAGGTTGAAAATG -3′
5′- AGTGGTTCCCAGAGTGAAACTGA -3′
5′- GCCAGAGCAAGGTTGAAAATG -3′
5′- GACATACCAGGACTGCCTGAGACAA -3′
5′- AGAACGAGATGTGGACAGCATGTTG -3′
5′- CGCTTCTGTGCCTGCTGCTCAT -3′
5′- AGCTTCCTTGGGACATTGGATGTTG -3′
5′- CCCTCTGCGCTCCTGCATCTG -3′
5′- GGAGCACTTGCCACTGGTGTAGA -3′
5′- ACACTCCAAGGGCCACCAGAA -3′
5′- AGGATGAAGGAGTCGATGCTGAT -3′
5′- CTGCTCAACCTGGCCATCTCT -3′
5′- CTTTTAAAGCAAACACAGCATGGAC -3′
5′- CAGGTCCTGCTGGAATCCAACATC -3′
5′- GTGCCGGCACCTGACACAGAAG -3′
5′- GGGCAAATACGTCATCTGTG -3′
5′- AGGGGATGACAAGGTCAAAG -3′
5′- CATGGCACCGTCAAGGCTGAGAA -3′
5′- CAGTGGACTCCACGACGTACTCA -3′
5′- GCTGCGGTAATCATGAGGATAAGA -3′
5′- TGAGCACAAGGCCTTCTAACCTTA -3′
5′- GCTGACCTGCTGGATTACATC -3′
5′- GAGAGATCATCTCCACCAATTAC -3′
5′- CTGGCACCACACCTTCTACAATGA -3′
5′- GCTGGGGTGTTGAAGGTCTCAAA -3′
5′- ATCCGCGCCATCATGGACAAGAA -3′
5′- TCGTCCTTCCGGGTATCAGTGAA -3′
5′- ACGGACCACCGCTGGAGCAA -3′
5′- TGCCATACTCCTCCCGGATAATC -3′
5′- ACTGGCCACTCGCTATTGCACA -3′
5′- TCCTCTATGCACAGTGCGATGACA -3′
5′- GCCAGTAGCCGTGCATACAGCTA -3′
5′- CGAGCAGTGATGCCTACCAACTG -3′
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Normalization genes
Average expression stability values of remaining control genes
Average expression stability M
0.6
0.55
0.5
0.45
0.4
0.35
0.3
0.25
0.2
HPRT1
HMBS
EIF2B2
<::::: Least stable genes
EEF2
ACTB
TBP
GAPDH
SDHA
Most stable genes ::::>
geNorm output of housekeeping gene stability across the samples from each subject group. RTPCR was performed as described in the Methods section with conditions consisting of an initial
hold step at 95°C for 10 min followed by 40 amplification cycles of 95°C for 5 s, 55°C for 10 s,
and 72°C for 20 s for the indicated genes. For sequences of the primer sets see Table,
Supplemental Digital Content 1. Ct values were transformed into relative quantification data
using the delta-Ct method followed by geNorm (version 3.4) analysis
(http://medgen.ugent.be/~jvdesomp/genorm/) [3]. The geNorm output chart ranking the
candidate reference genes according to their expression stability is shown.
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References for Supplemental Digital Content 1
1.
Gris P, Gauthier J, Cheng P, Gibson DG, Gris D, Laur O, et al. A novel alternatively
spliced isoform of the mu-opioid receptor: functional antagonism. Mol Pain 2010,6:33.
2.
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.
3.
Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, et al.
Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of
multiple internal control genes. Genome Biol 2002,3:RESEARCH0034.