Total RNA (20–30 μg) was isolated from HeLa or 293T cells cultured in a 90-mm Petri dish by using TRIzol Reagent. Small RNA fraction (containing U1, U2 snRNA and 5.8S rRNA) and large RNA fraction (containing 28S and 18S rRNA) were prepared from 10 μg of total RNA using mirVana miRNA Isolation Kit (Thermo Fisher Scientific AM1560). 28S or 18S rRNA was purified from large RNA fraction by reverse-phase LC as described in (44 (link)). Small RNA fraction was separated using 7% acrylamide denaturing urea PAGE and visualized with SYBR gold staining. RNAs corresponding to U1, U2 snRNA or 5.8S rRNA were excised, and eluted from PAGE gel piece by soaking with buffer containing 20 mM sodium acetate (pH 5.2). U1 or U2 snRNA purified from 10 μg of total RNA by reversed-phase LC on a PLRP-S 300Å column (2.1 × 100 mm, 3 μm, Agilent Technologies) was used for sequence-specific RNase H cleavage.
Plrp s 300 column
Manufactured by Agilent Technologies
The PLRP-S 300Å column is a reversed-phase high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of organic compounds. It features a porous polystyrene-divinylbenzene (PS-DVB) stationary phase with a pore size of 300 Angstroms, which makes it suitable for the analysis of medium to large molecular weight molecules.
Automatically generated - may contain errors
Lab products found in correlation
4000 column, by Agilent Technologies (2 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (1 mentions)
Thermocycler, by Beckman Coulter (1 mentions)
Sepasol rna 1 super g, by Nacalai Tesque (1 mentions)
Megascript t7 kit, by Thermo Fisher Scientific (1 mentions)
Rnase h, by Takara Bio (1 mentions)
Sybr gold, by Thermo Fisher Scientific (1 mentions)
Ultrafree mc, by Merck Group (1 mentions)
5 protocols using plrp s 300 column
1
Purification and Characterization of Small Nuclear RNAs
2
Oligonucleotide Purification and Annealing
3
rRNA Purification from Total RNA
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Total RNA (20 μg) was prepared from 10 ml TK6 cell culture (∼1.0 × 106 cell/ml) using Sepasol-RNA I Super G (Nacalai Tesque, Kyoto, Japan). rRNAs were purified from the total cellular RNA (∼15 μg) by reversed-phase LC on a PLRP-S 300Å column (2.1 × 100 mm, 3 μm, Agilent Technologies) or 4000 Å column (4.6 × 150 mm, 10 μm, Agilent Technologies) (28 (link)). A rRNA preparation of >95% purity was used for this study.
4
Synthetic RNA Production with Modified Nucleotides
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Each RNA was generated from its
corresponding DNA template digested with HindIII using an in vitro
transcription kit (MEGAscript T7 Kit, Thermo Fisher Scientific) in
the presence of m1ΨTP, ΨTP, or mo5UTP instead of uridine-5′-triphosphate (UTP) and/or in the
presence of m5CTP instead of cytidine-5′-triphosphate.
The 5′-capping structure was synthesized co-transcriptionally
using a cap 1 analog. The RNA was purified from the reaction (∼15
μg) by RPLC on a PLRP-S 300 Å column (2.1 × 150 mm,
3 μm; Agilent Technologies) or a 4000 Å column (4.6 ×
150 mm, 8 μm; Agilent Technologies)28 (link) and stored at −80 °C.
corresponding DNA template digested with HindIII using an in vitro
transcription kit (MEGAscript T7 Kit, Thermo Fisher Scientific) in
the presence of m1ΨTP, ΨTP, or mo5UTP instead of uridine-5′-triphosphate (UTP) and/or in the
presence of m5CTP instead of cytidine-5′-triphosphate.
The 5′-capping structure was synthesized co-transcriptionally
using a cap 1 analog. The RNA was purified from the reaction (∼15
μg) by RPLC on a PLRP-S 300 Å column (2.1 × 150 mm,
3 μm; Agilent Technologies) or a 4000 Å column (4.6 ×
150 mm, 8 μm; Agilent Technologies)28 (link) and stored at −80 °C.
5
Purification and Characterization of rRNA
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The purified rRNA (1 pmol) was digested with 5 U RNase H (Takara Bio) at 42°C for 1 h, guided by synthetic RNA/DNA hybrids complementary to the duplex cleavage sites (5 pmol, Supplemental Table 9 ) in 20 μL of 40 mM Tris–HCl (pH 7.7), 0.25 mM MgCl2, 1 mM DTT, and 4% glycerol. Before adding the enzyme, the sample was denatured at 65°C for 10 min. The reaction was stopped by adding 0.5 μL of 0.1 M EDTA, and the resulting fragments were separated by polyacrylamide gel containing 8 M urea. The gel was stained with SYBR Gold (Invitrogen) for 1 min, and the gel pieces containing RNA bands were excised from the gel and cut into small pieces. The RNA fragment was extracted by soaking the gel pieces in 80 μL of 20 mM triammonium citrate containing 4 M urea for 1 h. The extraction was carried out two times, and the extracts were combined and passed through a centrifugal filter unit equipped with a polyvinylidene fluoride membrane (Ultrafree-MC, Millipore). The RNAs in the eluate were finally purified by reversed-phase LC on a PLRP-S 300 Å column (2.1 × 100 mm, 3 μm, Agilent Technologies) as described previously (Yamauchi et al., 2013 (link); Taoka et al., 2010 (link)).
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