Generation of Full-Length cDNA Library from Single Human Prostate Cancer Cells

Several existing methods to construct cDNA libraries require several thousand cells and involve the lengthy procedure of reverse transcription (RT), restriction, adaptor ligation and vector cloning (5,6), which usually fail to maintain the completeness of a cDNA library, resulting in a loss of rare cDNAs. However, gene expression analysis of specific cell populations within a heterogeneous tissue is essential for research in vivo, requiring a method to generate cDNA libraries from a very small number of specific cells. The generation of amplified antisense RNA (aRNA) by incorporating an oligo(dT) primer coupled to a T7 RNA polymerase promoter sequence during RT has been developed to increase transcriptional copies of mRNAs from a limited amount of promoterlinked cDNAs (2,4). However, the aRNAs prepared from a single live neuron has been reported to cover 50%–75% of the total mRNA population (1,2), indicating that rare mRNAs were not protected during the amplification procedure. Here, we present a novel method for generating cDNA libraries that combines in situ RT (3) with amplified aRNAs followed by polymerase chain reaction (PCR) amplification. To protect mRNAs from degradation, the initial steps of in situ RT, 3′ oligonucleotide tailing and aRNA amplification are performed on fixed and permeabilized cells. Subsequent RT-PCR generates full-length cDNA libraries in a quantity easily manipulatable for gene expression analysis; the resultant cDNAs are amplified to total more than 109 the amount of original mRNAs. The method can be broken down into six steps (Figure 1, a–f): (a) cell fixation and permeabilization, (b) first RT, (c) terminal transferase tailing and cDNA double-stranding, (d) in-cell transcription, (e) second RT and (f) PCR amplification. Cell fixation and permeabilization (3). We have used LNCaP cells, cultured in RPMI 1640 medium (Irvine Scientific, Santa Ana, CA, USA) supplemented with 2% fetal calf serum, which were trypsinized and suspended in 1 mL of ice-cold 10% formaldehyde solution in 0.15 M NaCl. After a 1-h incubation on ice with occasional agitation, the cells were centrifuged at 11 000× g for 2 min and washed three times in ice-cold phosphate-buffered saline (PBS) with vigorous pipetting. The collected cells were permeabilized in 0.5% Nonidet P-40 (NP40; BDH Ltd., Poole, Dorset, England, UK) for 1 h with frequent agitation. After three washes in ice-cold PBS containing 0.1 M glycine, the cells were resuspended in 1 mL of the same buffer, aliquotted and stored at -70°C for up to a month. First RT. For RT of mRNAs in cells, twenty fixed cells were picked up under microscopy and suspended in 20 μL of ice-cooled diethyl pyrocarbonate (DEPC)-treated double-distilled (dd)H2O. An RT reaction (50 μL) was prepared, containing the above cold cells, 5 μL of 10× RT buffer [1.2 M KCl, 0.5 M Tris-HCl, 80 mM MgCl2, 50 mM dithiothreitol (DTT), pH 8.1 at 42°C], 25 pmol oligo(dT)-T7 primer (2), 0.2 mM of each dNTP and 80 U RNase inhibitor. After 6 U C. therm. Polymerase with Reverse Transcriptase Activity (Roche Molecular Biochemicals, Indianapolis, IN, USA) were added, the RT reaction was vortex mixed and incubated at 55°C for 2 min and then 68°C for 1 h. The cells were washed once with PBS and collected with an Amicon Microcon-50 Microconcentrator (Millipore, Bedford, MA, USA). Terminal transferase tailing and Benchmarks