The partial cDNA sequence was then used to make gene specific primers for a rapid amplification of cDNA ends (RACE) procedure that provided repeatable 5′ and 3′ cDNA ends.
Unexpectedly, the 3′-RACE procedure using the same primer led to amplification of a cDNA encoding a preprobradykinin whose signal peptide region was identical to that of preprotemporin-1SKa except for the substitution Ser18 → Asn.
Using a forward primer that was designed from a conserved region of the 5′-untranslated regions of Rana temporaria preprotemporins in a 3′-RACE procedure, a cDNA clone encoding preprotemporin-1SKa was prepared from R. sakuraii skin total RNA.
At the start of the race, all the cars lined up on the grid per FIA race procedure.
Prior to the RACE procedure, adapter-ligated cDNA was diluted fifty fold in TE-buffer and denatured by heating at 100°C for 2 min and thereafter placed directly on ice.
Using the 5'-RACE procedure with mRNA derived from the beta cell line βTC1 [24], we were unable to identify a discrete band corresponding to the start site of the endogenous pri-miR-375, presumably because of rapid processing of the precursor molecule [27].
The expression of SesB was also investigated with the 3'-RACE procedure.
The RLM-RACE procedure was successfully used to map the cleavage sites in five predicted va-miRNA target genes.
In order to experimentally confirm that AtPPC3 is a miR5640 target and to map the miR5640 cleavage site, we performed a modified RLM-RACE procedure [ 70].
In this study, the RLM-RACE procedure was successfully used to map the cleavage sites in four predicted target genes of C. trifoliata.
