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cDNA Libraries from Few Cells 499
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cDNA Libraries from a Low Amount of Cells
Philippe Ravassard, Christine Icard-Liepkalns, Jacques Mallet,
and Jean Baptiste Dumas Milne Edwards
1. Introduction
1.1. Application of the SLIC Strategy to cDNA Libraries:
General Considerations
Conventional cDNA library construction often requires a minimum available amount
of material (typically 1 or 2 µg of polyA + RNA). For complex organs, such as brain, or
certain species, such as humans, as well as subsets of cell types, this condition is often
difficult to fulfill. Amplification by polymerase chain reaction (PCR) can be used to
circumvent this limitation because it is a powerful method to obtain working quantities
of low-abundance DNAs. To effectively apply this method, known sequences need to
be attached to the ends of the single-stranded cDNA (ss-cDNA). One at the 5′ end of
the ss-cDNA is added during the priming of the synthesis; the other, at the 3′ end, is
covalently attached by ligation using the SLIC strategy. With known DNA sequences
attached to both ends of the synthetized cDNA, minute quantities can be amplified with
sequence-specific primers to provide sufficient material to successfully generate and
screen cDNA libraries. The overall scheme is illustrated in Fig. 1.
Obviously, the goal in constructing such a library is to maintain the representation
of every mRNA in the total RNA population, even low-abundant ones. Because the
reverse transcription and the single-strand ligation do not modify the overall proportion
of each molecule, only the PCR step may introduce an important bias in cDNA
representation. Thus, for cDNA library construction using this methodology, it is
important to optimize the synthesis of the sequence-tagged cDNA and to take steps
to limit any amplification bias.
1.2. Simultaneous PCR Amplification of a Complex DNA Mixture
Generates an Important Size Bias
The sequence-tagged cDNAs correspond to a large population of molecules with
indentical ends. The only difference between these molecules is their relative size and
sequence. Therefore, keeping the representativity of the cDNAs after PCR requires a
constant amplification yield regardless of the size and sequence of the original cDNA
population. This, unfortunately cannot be achieved by PCR. In fact, coamplification of
From: Methods in Molecular Biology, Vol. 226: PCR Protocols, Second Edition
Edited by: J. M. S. Bartlett and D. Stirling © Humana Press Inc., Totowa, NJ
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