Lna longrna gapmer

LNA™ longRNA GapmeRs (Exiqon, Denmark) are antisense oligonucleotides with perfect sequence complementary to their RNA target. The 2′C and 4′C atoms of these modified nucleotideare are linked by an oxymethylene bridge, the affinity of the LNA for target sequences increases and off-target effect significantly decreased^{45 (link),46 (link)}.
GapmeRs against n384546 were used for posttranscriptional lncRNA silencing. The sequences are shown in Table S3. Cells were transfected with 50 µM LNA longRNA GapmeRs with Lipofectamine 2000 reagent (Invitrogen, USA).

Bovine specific LNA™ longRNA GapmeRs (Exiqon, USA) were used to inhibit the expression of SMAD7 (5´-TTCGCAGAGTCGGCTA-3′ and 5´-CGATTTTGCTCCGTA-3′) ACVR2A (5´-GTTACTGGATTCGACG-3′ and 5´-GTTGGTCAGTAATCTA-3′). The transfection of 75 nM SMAD7 siRNA, ACVR2A siRNA or control siRNA was performed as described above. The gene expression analysis and cell proliferation assays were performed as described above.

LNA™ longRNA GapmeRs (antisense oligonucleotides (ASOs)) of 14–16 nucleotides) (Exiqon, Vedbaek, Denmark) are listed in Supplementary Materials. LNA™ longRNA GapmeRs or Scr control were reverse transfected at the indicated final concentrations and time points using Lipofectamine 2000 (Life Technologies) according to the manufacturer’s guidelines. Medium was changed 8 hours post transfection.

Antisense oligonucleotides (DNA-PS and 2′ OMe-PS gapmers), primers and probes were chemically synthesized using standard phosphoramidite chemistry. RNA oligonucleotides (21mer unmodified siRNAs and 27mer DsiRNAs) were chemically synthesized using t-Butyl-dimethylsilyl (TBDMS) chemistry (Integrated DNA Technologies). Probes for qPCR were purified using reversed phase high performance liquid chromatography (RP-HPLC) (Integrated DNA Technologies) while all other oligonucleotides were prepared as sodium salts. All oligonucleotides were analyzed by electrospray-ionization mass spectrometry (ESI-MS) and were within ±0.02% predicted mass. Oligonucleotide concentrations were calculated using modification-specific extinction coefficients based on measured ultraviolet (UV) absorbance at 260 nm. RNA duplexes were annealed in IDT duplex buffer (30 mM HEPES, pH 7.5, 100 mM Potassium Acetate) (Integrated DNA Technologies). Silencer^® Select siRNAs were purchased from Thermo Fisher Scientific (Waltham, MA, USA) and LNA™ longRNA GapmeRs were purchased from Exiqon (Vedbaek, Denmark). All oligonucleotide sequences are shown in Supplementary Table S1 in the online Supplemental Data.

The following LNA-longRNA GapmeRs (300600) from Exiqon were used to knock down the indicated genes: mSix1 #1 (CAAACTGGAGGTGAGT), mSix1 #2 (CAGAGGAGAGAGTTGA); mSox13 #1 (GCAAAGGCTGGTGGCT), mSox13 #2 (GAGGAGGAGGTTTAGC); mPim1 #1 (GGAGTTGATCTTGGAC), mPim1 #2 (GGTGATAAAGTCGA); mRreb1 #1 (GTTAGATTTGGTAGA), mRreb1 #2 (CGTTGATGAGAGGTG); Pparg (AGAAATCAACTGTGGT); scr (/56-FAM/AACACGTCTATACGC). Prior to transfection, the SVF cells were seeded on gelatinized 12 well plates (40,000 cells/well). On the next day, cells were transfected with 60 μM LNA-longRNA GapmerRs using 4.5 μl Lipofectamine 2000 (Invitrogen, 11668019).

LNA longRNA GapmeRs were ordered from Exiqon. The control gapmer (ACCagggcgtatctctccATA) [103] (link) and p21-specific gapmer (TCCgcgcccagCTCC) [77] (link) were administered to cells via gymnosis. The uppercase letters indicate the LNA while the lower case letters indicate phosphorothioated bases. On Day 0, the respective gapmer was added to the media for a final concentration of 100 nM. Cells were transfected, (as described above) on Day 2 and gapmer was readministered. On Day 4 (2 days post-transfection), cells were treated as described above for normal Sal transfection, with the addition of another dose of gapmer to the same final concentration.

HepG2 cells were maintained in Dulbecco's modified Eagle's medium, 10% FBS, and 100 units/mL penicillin streptomycin (Gibco) at 37°C, 5% CO₂. For the qPCR experiment, 80,000 cells were seeded in 12-well plates, and for the CAGE experiment, 160,000 cells were used in 6-well plates. Transfection was done according to the Lipofectamine RNAiMAX protocol. We used LNA longRNA GapmeRs by Exiqon, which are antisense oligonucleotides (ASOs) containing a central stretch of DNA monomers flanked by LNA blocks. Target RNAs are cleaved by RNase H, activated by ASOs. The ASOs were transfected into HepG2 cells at a concentration of 20 nM. Lipofectamine alone and scramble ASOs were used as controls. Knockdown efficiency was measured by qPCR with specific primer sets (Supplemental Table S10) for biological triplicates at 12, 24, 48, and 72 h after the transfection. GAPDH was used for normalization.