Construction and quality of cDNA libraries prepared from cytoplasmic RNA not enriched in poly(A)+RNA.
Poly(A)+RNA and cytoplasmic RNA of Ehrlich ascites tumor cells grown in vivo were used to study the quality and efficiency of cDNA synthesis. It was found that the rates of oligo(dT)-primed and unprimed reverse transcription were very similar in both cases. The size distributions of the cDNA strands prepared from unfractionated RNA reflected the size of cytoplasmic mRNA populations including a significant fraction of long molecules up to 6 kb. The fraction of cDNAs primed on rRNAs by oligo(dT) was found to be as low as 2-3%. Following second-strand synthesis by means of RNase H-induced nick translation by DNA polymerase I the overall yields in double-stranded cDNA were slightly higher when unfractionated cytoplasmic RNA was used as starting template. In repeated experiments we obtained an average yield of 2.2 micrograms of double-stranded cDNA when 70 micrograms of unfractionated cytoplasmic RNA was used as starting material. This amount of cDNA synthesized in one assay was sufficient to construct representative cDNA libraries in different vectors. Southern hybridizations of DNA isolated from cDNA libraries with various radiolabelled probes show that the libraries constructed from cDNA synthesized from cytoplasmic RNA not enriched in poly(A)+RNA contain a high ratio of full-length cDNA clones. The results suggest that representative cDNA libraries of high quality can be constructed without pre-isolation of poly(A)+RNA fractions.
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Institut für Zell- und Tumorbiologie, Deutsches Krebsforschungszentrum, Heidelberg, F.R.G.
Werner
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D000818:Animals; D015139:Blotting, Southern; D002286:Carcinoma, Ehrlich Tumor / Q000235:genetics; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis / Q000235:genetics*; D011061:Poly A / Q000235:genetics; D012313:RNA / Q000235:genetics*; D012333:RNA, Messenger; D012334:RNA, Neoplasm / Q000235:genetics
D012333:RNA, Messenger; D012334:RNA, Neoplasm; D011061:Poly A; D012313:RNA; D004247:DNA