Abstract
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.
aRNA amplification procedure.
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© 2003 Humana Press Inc., Totowa, NJ
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Lin, SL., Ji, H. (2003). cDNA Library Construction Using In Vitro Transcriptional Amplification. In: Ying, SY. (eds) Generation of cDNA Libraries. Methods in Molecular Biology™, vol 221. Humana Press. https://doi.org/10.1385/1-59259-359-3:93
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DOI: https://doi.org/10.1385/1-59259-359-3:93
Publisher Name: Humana Press
Print ISBN: 978-1-58829-066-3
Online ISBN: 978-1-59259-359-0
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