Subtraction hybridization cloning of RNA amplified from different cell populations microdissected from cryostat tissue sections.
We describe a generally applicable and easily reproducible method for the cloning of differentially expressed RNA, amplified from small numbers of enriched cell populations, obtained by microdissection from single cryostat sections. The procedure involves homopolymeric A tailing of cDNA synthesized from released RNA using an anchored (NN)T12 primer. Subsequent entire cDNA population polymerase chain reaction amplification was carried out using a biotinylated (X)nT16 primer-adaptor in the presence of biotin-dATP. This biotinylated driver cDNA was then twice hybridized in 50-fold excess to heterologous target cDNA made with nonbiotinylated (Y)nT16 primer; common hybrids and excess driver cDNA were magnetically removed following the addition of streptavidin-coated magnetospheres which bound biotinylated strands, leaving enriched target population sequences. These were then directly amplified through the tails using a primer containing only the target-specific (Y)n sequence. Insertion into a lambda-phage vector was facilitated by means of an EcoR1 site incorporated in the (Y)n primer. Subsequent packaging and transformation into Escherichia coli NM522 resulted in cDNA libraries containing approximately 5 x 10(3)-10(4) pfu. Screening of these primary libraries with cDNA derived from the starting populations yielded a large number of differentially hybridizing clones which are currently under analysis.
Luqmani
Y A
YA
Department of Medical Oncology, Charing Cross Hospital Medical School, London, United Kingdom.
Lymboura
M
M
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D016616:Cryoultramicrotomy; D017931:DNA Primers; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D012313:RNA / Q000235:genetics*
D017931:DNA Primers; D018076:DNA, Complementary; D012313:RNA