摘要
Background We constructed a cDNA subtractive library of dermal papilla cells (DPCs) in anagen with suppression subtractive hybridization (SSH) technique and clone differentially expressed genes related to DPCs in anagen. Methods Total mRNA was isolated from DPCs of anagen and telogen follicles. Moreover, single-strand (ss) and double-strand (ds) cDNAs were synthesized in turn using SMART PCR cDNA synthesis technology. ds cDNAs then were digested with Rsa I and divided into two groups, and ligated to the specific adaptor 1 and adaptor 2R, respect ively. After cDNAs were hybridized with each other twice and underwent two rounds of nested PCR. PCR products were ligated with arms of T/A plasmid vectors to set up the subtractive library. Selected clones were demonstrated by reverse Northern blot and sequenced. The acquired sequence data were aligned against the Genbank nucleotide database. Results cDNA subtractive library of DPCs in anagen follicles was set up successfully with high subtractive efficiency. Thirty-five genes were identified in this study with 22 known functional genes and 13 unknown functional genes. Conclusions All results confirm the effectiveness and sensitivity of SSH in detecting differentially expressed genes from a small amount of clinical samples. Information about such alterations in gene expression could be useful for elucidating the genetic events in hair follicle growth regulation.
Background We constructed a cDNA subtractive library of dermal papilla cells (DPCs) in anagen with suppression subtractive hybridization (SSH) technique and clone differentially expressed genes related to DPCs in anagen. Methods Total mRNA was isolated from DPCs of anagen and telogen follicles. Moreover, single-strand (ss) and double-strand (ds) cDNAs were synthesized in turn using SMART PCR cDNA synthesis technology. ds cDNAs then were digested with Rsa I and divided into two groups, and ligated to the specific adaptor 1 and adaptor 2R, respect ively. After cDNAs were hybridized with each other twice and underwent two rounds of nested PCR. PCR products were ligated with arms of T/A plasmid vectors to set up the subtractive library. Selected clones were demonstrated by reverse Northern blot and sequenced. The acquired sequence data were aligned against the Genbank nucleotide database. Results cDNA subtractive library of DPCs in anagen follicles was set up successfully with high subtractive efficiency. Thirty-five genes were identified in this study with 22 known functional genes and 13 unknown functional genes. Conclusions All results confirm the effectiveness and sensitivity of SSH in detecting differentially expressed genes from a small amount of clinical samples. Information about such alterations in gene expression could be useful for elucidating the genetic events in hair follicle growth regulation.
基金
ThisworkwassupportedbytheNationalNaturalScienceFoundationofChina (No 3 0 0 70 3 75)
