( Generate cDNA libraries with a...)Constructing Directional cDNA Libraries from Limited Amounts of RNA

Generate cDNA libraries with as little as 50 ng of RNA

Barry R. Neiditch Cherie Dewar Tanya Hosfield Marie Callahan
John C. Bauer Mike Kobrin Quinn Lu Dianne Eckhardt
Stratagene

Melanie Lenhart Paul Young
Human Genome Sciences, Rockville, Maryland

Stratagene has developed the PCR Library Construction Kit, which introduces a new method to generate high-efficiency cDNA libraries in a plasmid vector. Use this kit with either enriched poly(A) ⁺ RNA or as little as 50 ng of total RNA (starting material); this minuscule amount compares well with the 5 to 10 g of RNA required when using conventional methods. The kit is based on an approach that combines linker-mediated polymerase chain reaction (PCR) for amplifying cDNA and ligation-independent cloning (LIC) for cloning PCR products. We used the kit to create an ovarian cancer cDNA library from tissue isolated by laser-capture microdissection. To evaluate our PCR methodology, we compared HepG2 libraries constructed using both traditional lambda techniques and our PCR method. Although a shift occurred in the average insert size, sequence analysis revealed that the distribution of gene classes was comparable between both libraries. Moreover, sequencing confirmed that in the PCR-generated library, no gene was over represented due to artifacts; hence, our technique is ideal for generating libraries when starting with a limited amount of material.

While cDNA libraries have become standard tools for gene discovery and characterization, conventional libraries suffer limitations. In order to achieve libraries of significant complexity, relatively large amounts of poly (A)⁺ RNA (5 to 10 g) are required. To bypass this requirement, attempts have been made to apply PCR technology to the synthesis of libraries from small cell populations. Unfortunately, these
'"/>

Source: