Supplementary Methods
Synthesis of antagomirs
RNAs were synthesized using commercially available 5'-O-(4,4'-dimethoxytrityl)-2’-Omethyl-3'-O-(2-cyanoethyl-N,N-diisopropyl) RNA phosphoramidite monomers of 6-Nbenzoyladenosine (ABz), 4-N-benzoylcytidine (CBz), 2-N-isobutyrylguanosine (GiBu), and
uridine (U), according to standard solid phase oligonucleotide synthesis protocols1. For
antagomirs. i.e., cholesterol conjugated RNAs, the synthesis started from a controlledpore glass solid support carrying a cholesterol- hydroxyprolinol linker2. Antagomirs with
phosphorothioate backbone at a given position were achieved by oxidation of phosphite
with phenylacetyl disulfide (PADS) during oligonucleotide synthesis3. After cleavage and
de-protection, antagomirs were purified by reverse-phase high-performance liquid
chromatography, while the unconjugated RNA oligonucleotides were purified by anionexchange high-performance liquid chromatography. Purified oligonucleotides were
characterized by ES mass spectrometry and capillary gel electrophoresis.
Gene expression analysis
Total RNA from liver of mice treated with antagomirs was isolated three days after
treatment. RNA was pooled from 3 animals for each group. The integrity of the RNA
samples was assessed by denaturing formaldehyde gel analysis. First strand cDNA
synthesis was completed with total RNA (10 µg) cleaned with RNAeasy columns
(Quiagen) and the Superscript double stranded cDNA synthesis protocol (Invitrogen),
except that HPLC purified T7-promoter-dT30 primer (Genset) was used to initiate the
first strand reaction. Biotin labeled cRNA was synthesized from T7 cDNA using the
RNA transcript labeling kit (Enzo). The sample was cleaned with the Affymetrix
GeneChip sample clean up module (Qiagen) to remove free nucleotides and quantitated
spectrophotometrically. Biotin-labeled cRNA was fragmented and hybridized to mouse
genome 430 2.0 arrays according to the manufacturer’s manual with a final concentration
of fragmented cRNA of 0.05 µg/µl. The arrays were scanned using a Hewlett Packard
confocal laser scanner and analyzed using ArrayAssist Lite and Affymetrix® Microarray
1
Suite version 5.0 software. The p-value for comparison of each probe set was computed
using the Wilcoxon's Signed Rank test.
RT-PCR
Extraction of total RNA, synthesis of cDNA, and PCR were carried out as described
previously4. Total RNA was extracted from tissues and contaminating genomic DNA was
removed by treating with 5 u of RNase-free DNase-I (Ambion)/10 µg of RNA. cDNA
was synthesized using the first-strand Superscript cDNA synthesis kit and random
hexamer primers (Invitrogen). The cDNAs provided templates for polymerase chain
reactions (PCRs) using specific primers at annealing temperatures ranging between 60
and 65C in the presence of dNTPs, [-32P]dCTP, and Taq DNA polymerase. The primer
sequences used for PCR are available upon request.
Gene Ontology analysis
Refseq identifiers were mapped to MGI identifiers using a map provided by ensembl
(http://www.ensembl.org/Multi/martview). We then used the program FuncAssociate5
(http://llama.med.harvard.edu/cgi/func/funcassociate) with default settings to search for
overrepresented Gene Ontology terms. Results were sorted by LOD scores.
Independently, we obtained very similar results with the program GeneMerge v.1.2 and
applying a conservative Bonferroni correction for multiple testing6.
HMG-CoA reductase (Hmgcr) activity assay
Hepatic microsomal HMGCR activity was assayed by a method modified from a
previously published procedure7. Briefly, 75 g of hepatic microsomal protein extracts
were preincubated with an NADPH-generating system (3.4 mM NADP+, 30 mM glucose
6-phosphate, 1 unit of glucose-6-phosphate dehydrogenase) in buffer (50 mM K2HPO4,
70 mM KCl, 10 mM DTT, 30 mM EDTA, 50mM NaF, pH 7.5). The reaction was started
with the addition of 15 µl
14
C-labeled substrate (160 M [final concentration] HMG-
CoA/[14C]HMG-CoA, 0.06 Ci, Amersham). The mixture was incubated for 30 min and
stopped with 15 µl 6 M HCl. [3H]mevalonolactone and unlabeled mevalonolactone were
2
added for recovery of standard and product marker, respectively. After lactonization the
products were briefly centrifuged, supernatants dried and dissolved in acetone. Samples
were
separated
by
TLC
on
Silica
Gel
60 plates
(VWR
Scientific)
with
acetone/toluene/acetic acid (75:25:1, vol/vol) as the solvent system. The immediate
product (14C-labeled mevalonolactone) was quantitated by scintillation spectrometry.
References:
1. Damha, M. J. & Ogilvie, K. K. Oligoribonucleotide synthesis. The silyl-phosphoramidite method. Methods Mol. Biol. 20, 81–114 (1993).
2. Manoharan, M., Kesavan, V. & Rajeev, K. G. SiRNA’s containing ribose substitutes to
which lipophilic moieties may be attached. US patent application Publ. 2005, 273 pp. US
2005107325 A1 20050519
3. Cheruvallath, Z. S., Wheeler, P. D., Cole, D. L. & Ravikumar, V. T. Use of
phenylacetyl disulfide (PADS) in the synthesis of oligodeoxyribonucleotide
phosphorothioates. Nucleosides Nucleotides 18, 485–492 (1999).
4. Shih, D. Q. et al. Profound defects in pancreatic beta-cell function in mice with
combined heterozygous mutations in Pdx-1, Hnf-1alpha, and Hnf-3beta. Proc. Natl Acad.
Sci. USA 99, 3818–3823 (2002).
5. Berriz, G. F., King, O. D., Bryant, B., Sander, C. & Roth, F. P. Characterizing gene
sets with FuncAssociate. Bioinformatics 19, 2502–2504 (2003).
6. Castillo-Davis, C. I. & Hartl, D. L. GeneMerge–post-genomic analysis, data mining,
and hypothesis testing. Bioinformatics 19, 891–892 (2003).
7. Nguyen, L. B. et al. J. Molecular defect in hepatic cholesterol biosynthesis in
sitosterolemia with xanthomatosis. Clin. Invest. 86, 923–931 (1990).
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