Suppression subtractive hybridization as a tool to identify anthocyanin metabolism-related genes in apple skin.
The pigmentation of anthocyanins is one of the important determinants for consumer preference and marketability in horticultural crops such as fruits and flowers. To elucidate the mechanisms underlying the physiological process leading to the pigmentation of anthocyanins, identification of the genes differentially expressed in response to anthocyanin accumulation is a useful strategy. Currently, microarrays have been widely used to isolate differentially expressed genes. However, the use of microarrays is limited by its high cost of special apparatus and materials. Therefore, availability of microarrays is limited and does not come into common use at present. Suppression subtractive hybridization (SSH) is an alternative tool that has been widely used to identify differentially expressed genes due to its easy handling and relatively low cost. This chapter describes the procedures for SSH, including RNA extraction from polysaccharides and polyphenol-rich samples, poly(A)+ RNA purification, evaluation of subtraction efficiency, and differential screening using reverse northern in apple skin.
Ban
Yusuke
Y
Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan.
Moriguchi
Takaya
T
D000872:Anthocyanins / Q000378:metabolism*; D015723:Gene Library; D017343:Genes, Plant / Q000235:genetics*; D027845:Malus / Q000235:genetics* / Q000378:metabolism* / Q000528:radiation effects; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015341:Nucleic Acid Probes / Q000235:genetics; D019441:Plant Epidermis / Q000235:genetics* / Q000378:metabolism* / Q000528:radiation effects; D016133:Polymerase Chain Reaction; D011062:Polynucleotide Adenylyltransferase / Q000378:metabolism; D018749:RNA, Plant / Q000235:genetics / Q000302:isolation & purification; D015203:Reproducibility of Results; D014466:Ultraviolet Rays
D000872:Anthocyanins; D015341:Nucleic Acid Probes; D018749:RNA, Plant; D011062:Polynucleotide Adenylyltransferase