1-4. Experimental Procedures
I) Isolation of RNA Experimental Procedures
Cytoplasmic RNA and Poly A+ RNA were isolated according to the standard method. Oligo-dT cellulose was from Collaborative Biomedical Products and Roche.
  
II) the oligo-capping procedure Experimental Procedures
Oligo-capping was performed as described (Maruyama and Sugano, 1994), with some modifications. In brief, 10 to 50 ug of polyA+ RNA was treated with 1.2 units of bacterial alkaline phosphatase (BAP; TaKaRa) in 100ul of 100mM Tris-HCl (pH 8.0), 5mM 2-mercaptoethanol with 100 units of RNasin (Promega) at 37oC for 60 min. After extraction with phenol:chloroform (1:1) ethanol precipitation, the polyA+ RNA was treated with 20 units of tobacco acid pyrophosphatase (TAP) in 100ul of 50mM sodium acetate (pH 5.5), 1mM EDTA, 5mM 2-mercaptoethanol with 100 units of RNasin at 37oC for 60 min. After phenol:chloroform extraction and ethanol precipitation, 2 to 4ug of the BAP-TAP treated polyA+ RNA were ligated with 0.4ug of 5'-oligo (KM-02; 5'-AGC AUC GAG UCG GCC UUG UUG GCC UAC UGG-3') using 250 units of RNA ligase (TaKaRa) in 100ul of 50mM Tris-HCl (pH7.5), 5mM MgCl2, 5mM 2-mercaptoethanol, 0.5mM ATP, 25% PEG8000 with 100 units of RNasin at 20oC for 3 hours.
III) the first strand cDNA synthesis Experimental Procedures
After removing unligated 5'-oligo, cDNA was synthesized with RNaseH free reverse-transcriptase (Superscript II, Gibco BRL). 10pmol of dT adapter-primer (5'-GCG GCT GAA GAC GGC CTA TGT GGC CTT TTT TTT TTT TTT TTT-3') was used in 50ul with 2 to 4ug of oligo-capped polyA+ RNA. The reaction conditions were as recommended by the supplier and incubated at 42oC for 3 hour.
IV) cDNA amplification Experimental Procedures
  After first strand synthesis, RNA was degraded in 15 mM NaOH by incubating at 65oC for 1 hour. The cDNA which is made from "oligo-capped" polyA+ RNA was amplified in a volume of 100 ul using an XL PCR kit (Perkin-Elmer) with 16 pmol of 5' (5'-AGC ATC GAG TCG GCC TTG TTG-3') and 3' (5'-GCG GCT GAA GAC GGC CTA TGT-3') PCR primers. For dT-adapter primer primed cDNA, amplification cycles were 12 cycles at 94 oC for 1 min, 58 oC for 1 min, and 72 oC for 10 min. PCR products were extracted with phenol:chloroform (1:1) once, ethanol precipitated and digested with SfiI. SfiI-digested PCR products were separated by an agarose gel electrophoresis and products longer than 1 kb were isolated and cloned into DraIII-digested pME18S-FL3. In this way, the cDNA could be cloned into the vector in an orientation-defined manner.
V) Sequencing Experimental Procedures
  Plasmid DNA was isolated using PI-100 and PI-200 auto-plasmid-isolators (KURABO). Sequences were determined by the dideoxy termination method using BigDye sequencing kit (ABI). The sequence was read by ABI 3700 (ABI) auto-sequencers.
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