A multiplex reverse transcriptase-polymerase chain reaction method for fluorescence-based semiautomated detection of gene expression in Arabidopsis thaliana.
A non-radioactive, rapid and sensitive method is presented for the simultaneous detection of several mRNA molecules. The technique is based on conventional first-strand cDNA synthesis by reverse transcriptase, followed by multiplex polymerase chain reaction (PCR) co-amplification of several gene products in a reaction mix containing several primer sets, each including a fluorescently labeled oligonucleotide. The PCR products obtained are finally electrophoresed in a single lane of a polyacrylamide gel, in an automated DNA sequencer controlled by fragment-analysis software. The method has proven useful to efficiently detect nine mRNA transcripts, some of which are low copy number, from small specimens such as single flowers and leaves of Arabidopsis thaliana (L.) Heynh. This approach might be easily extended to other biological systems, for developmental and physiological analyses, population studies and diagnosis.
Ponce
M R
MR
División de Genética, Universidad Miguel Hernández, Alicante, Spain.
Pérez-Pérez
J M
JM
Piqueras
P
P
Micol
J L
JL
D017360:Arabidopsis / Q000235:genetics*; D001331:Automation; D001483:Base Sequence; D017931:DNA Primers; D005453:Fluorescence; D015870:Gene Expression; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers