The mRAP protocol and its application to Jurkat cells. (A) Isolated Small-RNA molecules are ligated to the 3′ adaptor (3′-ADP) and subjected to reverse transcription with the RT primer. After annealing of the 5′ adaptor (5′-ADP) to the poly(C) overhang at the 3′ end of the synthesized cDNAs, the latter are subjected to PCR with the 5′ and 3′ PCR primers. After an extensive cloning/sequencing of the PCR products, we noticed that, of the three major sizes of amplicon generated, only the middle one includes cDNAs derived from miRNAs. The large product of ∼120 bp is composed of two 5′ adaptors and one 3′ adaptor without miRNA sequences. The small product of ∼70 bp is, on the other hand, composed of only one 5′ adaptor and one 3′ adaptor. The product of ∼90 bp are thus isolated, digested with BanI, and self-ligated to yield concatamers. (B) Among 1652 mRAP clones of Jurkat cells that matched the human genome sequence, 616 clones corresponded to known miRNAs, 17 are candidates for novel miRNAs and 219 corresponded to rRNAs, 166 to tRNAs, 127 to transposable elements, 112 to simple repeats and 395 to other genomic sequences that do not fold into a hairpin or otherwise fail the miRNA prediction pipeline. (C) Alignment of the nucleotide sequence (red) of one predicted novel miRNA (Hsj_43) with genomic sequences of human, chimpanzee, dog, mouse, cow, rat and chicken. Nucleotides conserved between human and other species are shaded in gray. Possible base pairing schemes for the respective Hsj_43 precursors are shown below the aligned sequences and, for the human sequence, in the upper inset.