Identification of microRNAs and other tiny noncoding RNAs by cDNA cloning.
MicroRNAs (miRNAs) and other small RNAs can be identified by cloning and sequencing cDNAs prepared from the approximately 22-nt fraction of total RNA. Methods are described for the construction of cDNA libraries from small noncoding RNAs through the use of T4 RNA ligase, reverse transcriptase, and polymerase chain reaction. cDNAs are cloned in lambda or plasmid vectors, and the sequences are compared to annotated genomic sequence databases, and analyzed by RNA folding programs to distinguish miRNA sequences from other small RNAs of similar size. Northern blot hybridization is used to confirm the expression of small RNAs in vivo.
Ambros
Victor
V
Department of Genetics, Dartmouth Medical School, Hanover, NH, USA.
Lee
Rosalind C
RC
D020413:3' Untranslated Regions / Q000302:isolation & purification; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D007202:Indicators and Reagents; D035683:MicroRNAs / Q000235:genetics* / Q000302:isolation & purification; D022661:RNA, Untranslated / Q000235:genetics / Q000302:isolation & purification*; D013698:Templates, Genetic
D020413:3' Untranslated Regions; D017931:DNA Primers; D018076:DNA, Complementary; D007202:Indicators and Reagents; D035683:MicroRNAs; D022661:RNA, Untranslated