GLGI技术初步验证系统性红斑狼疮患者CD4^+、CD8^+T细胞基因表达谱

Identification of gene profiles of CD4^+ and CD8^+ T lymphocyts in systemic lupus erythematosus by generation of longer cDNA fragments from serial analysis of gene expression tags for gene identification

目的研究GLGI技术鉴定系统性红斑狼疮患者LongSAGE标签的方法。方法运用GLGI技术,将包括17 bp的SAGE标签及单一碱基和oligo(dT)锚定引物进行PCR扩增,得到表达基因的3′端序列,并确认表达基因。结果单一匹配标签12个有9个获得有效扩增,包括原来的17 bp的SAGE标签序列,其长片段测序后有＞85%的碱基与已知基因相符合,表明为该已知基因。20个无匹配标签中未发现有新的基因。结论GLGI技术能够有效识别基因,LongSAGE-GLGI技术是鉴定SAGE大规模标签库较好的方法。

Objective To identify LongSAGE Tags in systemic lupus erythematosus （SLE） by generation of longer cDNA fragments from serial analysis of gene expression （ SAGE ） tags for gene identification （ GLGI ）. Methods CD4^＋ and CD8^＋ T lymphocytes were collected from the PBMCs of 25 patients with SLE and 10 healthy controls. Then the total RNA was extracted and reverse transcribed to cDNA. After that, PCR was performed with the cDNA as plate, a 17-base tag as the sense primer, and a single base anchored oligo （ dT ） as the antisense primer. With the GLGI approach, the longSAGE tags were converted into their corresponding 3′ end of cDNA fragments covering hundreds of bases, which were identified by DNA cloning and sequencing. Results Nine of twelve tags matching a specific single gene were stably and efficiently extended, including the primary 17-bp SAGE tag sequence. And their long fragments had a consistency of more than 85% with the corresponding known sequences, which suggested that these tags originated from the known genes. No new genes were found in 20 non-matching genes. Conclusion GLGI technology can efficiently identify the genes from LongSAGE, and the combined application of LongSAGE and GLGI is a good method for large-scale identification of SAGE tags.