Multiple isoform recovery (MIR)-PCR: a simple method for the isolation of related mRNA isoforms.
We present a rapid and efficient method for the detection of related transcripts with different expression levels. This approach combines the rapid amplification of cDNA ends (RACE) method with a cDNA subtractive technique. The strategy is based on successive subtractions of prevalent isoforms resulting in enrichment of less expressed transcripts. For each subtraction, a biotinylated primer specific for the prevalent isoform is hybridized on the total cDNA and the hybrid is retained on a streptavidin affinity column. The unbound cDNA serves as a template for subsequent isoform identification. To illustrate its application we describe the isolation of three new actin cDNA isoforms in the freshwater planarian Dugesia (S) polychroa.
Fagotti
A
A
Institute of Comparative Anatomy, University of Perugia, Perugia, Italy.
Gabbiani
G
G
Pascolini
R
R
Neuville
P
P
D000199:Actins / Q000096:biosynthesis* / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D018076:DNA, Complementary; D008969:Molecular Sequence Data; D010932:Planarians / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000302:isolation & purification*
D000199:Actins; D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger