Construction of cDNA Libraries from Small Quantities of Total RNA Using Template Switching Catalyzed by M-MLV Reverse Transcriptase

Abstract

Generation of high quality cDNA libraries with a comprehensive representation of the original mRNA population requires relatively large amounts (5–50 μg) of poly ( A)⁺RNA which is difficult to obtain when the amount of biological material is limited ( e.g., rare and unstable cell lines, microdissected cancer cells, biopsy materials, pathological specimens, embryonic and neuron tissues, cells in body fluids and so on). To circumvent this problem, several PCR-based technologies for amplification of cDNA from small amounts of total RNA have been described (Froussard 1993; Bertioli et al. 1994; Korneev et al. 1994). Basically, the amplification of a total cDNA population requires that universal primer binding sites are available at both cDNA ends. An arbitrary sequence can easily be imposed at the 5′ cDNA end by priming reverse transcription from the poly (A)⁺RNA fraction of total RNA by oligo (dT) or anchored oligo (dT) primer. Several strategies have been developed to add a determined sequence (anchor) at the 3′ end of the first-strand cDNA. These strategies include: (1) oligo (dG) or oligo (dA) tailing by terminal deoxynu- cleotidyltransferase (Bertioli et al. 1994; Korneev et al. 1994); ( 2) the use of T4 RNA ligase to covalently attach a single-stranded (ss) anchor oligonucleotide to the 3′ end of the ss cDNA (Apte and Siebert 1993); (3) the ligation of double-stranded (ds) adaptors to both ends of the ds cDNA (Frohman et al. 1988); and (4) the removal of the 7-MeGppp cap structure followed by ligation of an anchor sequence to the 5′ end of the decapped mRNA by T4 RNA ligase (Fromont-Racine et al. 1993).