A REVERSE TRANSCRIPTASE-POLYMERASE CHAIN-REACTION ASSAY FOR GENE-EXPRESSION STUDIES AT THE SINGLE-CELL LEVEL

In order to investigate gene expression during events before and after fertilization of maize and other higher plants, a very sensitive tech nique is required. Plant material is very limited and, therefore, conv entional Northern blots cannot be performed. The reverse transcriptase -polymerase chain reaction (RT-PCR) technique is a highly sensitive me thod for detecting RNA transcripts in small samples of cells or tissue s which employs reverse transcription of the target RNA followed by PC R amplification of its cDNA. In order to detect exactly when and in wh ich type of cell genes are expressed during very early embryogenesis o f maize, from gametes to the zygote, we developed a RT-PCR technique w hich is reliable and reproducible for single cell assays. A glyceralde hyde-3-phosphate dehydrogenase (GAPDH) gene from maize has been used a s a model gene for the development of the RT-PCR detection method. In addition, a tissue specific gene of the MCM2/3/5 gene family, expresse d at very low levels, was analysed to confirm the reliability of the m ethod. The protocol described here has been optimized for single cells from different cell types of maize and other cereals and is, to our k nowledge, the first report of RT-PCR with isolated single cells from p lants.