cDNA Library Construction Using In Vitro Transcriptional Amplification

The generation of a complementary DNA (cDNA) library using transcriptional amplification has been developed to offer better linear amplification than polymerase chain reaction (PCR)-based methods. By incorporating a RNA promoter element during reverse transcription of messenger RNA (mRNA), a cDNA library can be preserved and amplified in the form of antisense RNA (aRNA) construct. In brief, the aRNA amplification procedure (see Fig. 1 ) is based on (1) reverse transcription of poly(A)⁺ RNAs with promoter-linked oligo-(dT) primers, (2) double-stranding the resulting cDNA, (3) vitro transcription from the promoter element of the cDNA to synthesize the aRNA sequence up to 2000-fold increase, (4) reverse transcription of the aRNA, (5) denaturation and then double-stranding the resulting cDNA with promoter-linked oligo-(dT) primers, and (6) repeating steps 3–5 to achieve the desired cDNA or aRNA amount for the library preparation.

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