( e pairs ( Figure 3 ). Sequen...)Constructing Directional cDNA Libraries from Limited Amounts of RNA

Fig.3

With the kit, we successfully generated other cDNA libraries as well. These libraries were constructed from 50 to 100 ng of total RNA from human macrophage cells, human prostate cells, and several cancerous tissues derived from LCM. All libraries contained a low percentage of nonrecombinants, and inserts were confirmed to contain polyadenlylation sequences.

Sequence Analysis and Comparison of HepG2 Libraries

We compared the sequence analysis of two HepG2 libraries: A HepG2 Lambda ZAP II library was constructed starting with 5 g of poly (A)⁺ RNA using traditional methods, and a second HepG2 library was created with 100 ng of total RNA using the PCR-based method previously described. In each case, over 1000 clones were analyzed, then the clones were characterized into several classes (Table 1). The average insert size shifted from 2.1 kb in the lambda library to 0.8 kb in the PCR library. In Table 1, the breakdown of analyzed sequences into gene classes is described for each library, and the known genes identified for both libraries coordinate with the expected sequences that we observed to be expressed in liver tissue.

Table 1: Breakdown of Sequencing Results by Class

Class 1: Sequence is identical to a known human gene
Class 2: Sequence has a significant match with a human protein
Class 3: Sequence has a significant match to a nonhuman protein
Class 4: Mitochondrial and vector sequence
Class 5: Unknown genes. Sequence has no significant match in the public database