Visualization of gene expression by fluorescent multiplex reverse transcriptase-PCR amplification.
Many developmental and physiological analyses, population studies, and diagnostic tests can be performed by simply determining the presence or absence of a limited number of gene products. Here, we describe a rapid and sensitive procedure, based on the reverse transcription of total RNA samples followed by the co-amplification of specific complementary DNA molecules, for the simultaneous detection of different transcripts. Multiplex PCR amplification products are obtained in a single reaction mix containing several primer pairs, each of which includes a fluorescently labeled oligonucleotide; the amplification products are finally electrophoresed in an automated DNA sequencer controlled by fragment analysis software. The electropherograms obtained in this way allow a semiquantitative and efficient visualization of gene expression.
Ponce
María Rosa
MR
División de Genética and Instituto de Bioingeniería, Universidad Miguel Hernández, Alicante, Spain.
Quesada
Víctor
V
Hricová
Andrea
A
Micol
José Luis
JL
D017360:Arabidopsis / Q000235:genetics; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D005456:Fluorescent Dyes; D015870:Gene Expression; D017343:Genes, Plant; D018749:RNA, Plant / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014158:Transcription, Genetic
D017931:DNA Primers; D018744:DNA, Plant; D005456:Fluorescent Dyes; D018749:RNA, Plant