![]() Quantitative PCR was used to determine the relative abundance of internally primed cDNA and 3′ primed cDNA extending beyond the internal poly(A) tract ( 12). Four micrograms of total RNA from CD15+ myeloid progenitor cells was used for reverse transcription with either 200 ng oligo(dT) primer or 200 ng of the set of anchored oligo(dT) primers as described above. Selection of mRNAs of Known Genes with Internal Poly(A) Sequences for Reverse Transcription and Quantitative PCR.Ī group of mRNA sequences, each containing a minimum of eight continuous internal As, were selected from the genes expressed in CD15+ myeloid progenitor cells ( 11). Sequences were collected with an ABI377 DNA sequencer (Applied Biosystems). Clones were picked randomly and the insert in each clone was amplified with T7 primer located in the vector and T3 primer located in the insert, and used for sequencing reactions with T7 primer and a Big-Dye sequencing kit (Applied Biosystems). The resulting single-strand cDNA was converted into double-strand cDNA by PCR with M13F primer (5′-GTAAAACGACGGCCAGTACG-3′) and antisense primer (5′-ACTATCTAGAGCGGCCGCTT-3′) and cloned into TOPO TA vectors (Invitrogen). Reverse transcription was performed by use of a cDNA synthesis kit containing Moloney murine leukemia virus reverse transcriptase (Life Technologies). The primers used for priming were either the regular oligo dT16 (5′-ACTATCTAGAGCGGCCGCTTT16–3′) (200 ng per reaction) or a mixture of five anchored oligo dT16 primers (5′-ACTATCTAGAGCGGCCGCTTT16-A, -G, -CA, -CG, and -CC-3′) ( 10) (200 ng per reaction). ![]() One microgram of in vitro transcript was used as the template for reverse transcription. We also demonstrated that the problem of internal poly(A) priming could be minimized by simple replacement of the oligo(dT) primer with a set of anchored oligo(dT) primers for cDNA synthesis. Our studies reveal the patterns of internal poly(A) priming and the prevalence of the resulting truncated cDNAs in the public EST database. We performed a series of experiments and analyzed the current databases to investigate systematically the issue of internal poly(A) priming by oligo(dT) primer. The occurrence of 3′-truncated ESTs related to internal poly(A) was observed in earlier studies based on limited data, with estimated rate of 2.5–14% of the ESTs ( 5, 9). We speculated that these internal poly(A) sequences could also be primed by oligo(dT) primers during reverse transcription, which would result in the generation of two kinds of truncated cDNA sequences: one starting from the internal poly(A), the other starting from the 3′ poly(A), but terminated before the internal poly(A) sequence (Fig. Besides being located universally at the 3′ end of mRNA, poly(A) sequences can also be located within mRNA sequences.
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