Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100193, China.
Mol Biol Rep. 2012 May;39(5):5905-12. doi: 10.1007/s11033-011-1402-1. Epub 2011 Dec 27.
Accurate quantification of transcript profiling with quantitative real time polymerase chain reaction (qRT-PCR) relies on the reliable normalization of an appropriate reference gene. This study reported the identification and validation of nine reference genes, including β-tubulin (β-TUB), elongation factor 1 alpha (EF-1α), elongation factor 1 beta (EF-1β), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin (UBQ), actin 1/2(ACT-1 and ACT-2), 18S rRNA, and 26S rRNA, from Anoectochilus roxburghii (Wall.) Lindl., a valuable herb remedy widely used for various diseases treatment in traditional Chinese medicine. Transcriptional levels of the candidate reference genes were examined using qRT-PCR analysis and revealed differential expression of the genes in the leaf, stem, root, flower, and peduncle tissues. The relative quantities data were subjected to geNorm software for ranking the expression stability of the reference genes and the results showed that EF-1β and ACT-2 were the two best stable genes whereas GAPDH and 26S rRNA did not favor normalization of qRT-PCR in these tissues. The expression pattern of a squalene synthase encoding gene (SS) was also determined in parallel. The analyses were in great consistency when the qRT-PCR data was normalized to the expression of each or both of EF-1β and ACT-2 as the internal control, further confirming the reliability of EF-1β and ACT-2 as the best internal control. The present study provided the first important clues for accurate data normalization in transcript profiling in A. roxburghii, which will be essential to further functional genomics study in the valuable medicinal plant.
实时荧光定量聚合酶链反应（qRT-PCR）准确量化转录谱依赖于合适的参考基因的可靠归一化。本研究报道了从铁皮石斛（Wall.）Lindl.中鉴定和验证了 9 个参考基因，包括β-微管蛋白（β-TUB）、延伸因子 1α（EF-1α）、延伸因子 1β（EF-1β）、甘油醛-3-磷酸脱氢酶（GAPDH）、泛素（UBQ）、肌动蛋白 1/2（ACT-1 和 ACT-2）、18S rRNA 和 26S rRNA，铁皮石斛是一种有价值的草药，广泛用于中药治疗各种疾病。使用 qRT-PCR 分析检测候选参考基因的转录水平，并显示基因在叶、茎、根、花和花梗组织中的差异表达。相对数量数据通过 geNorm 软件进行分析，以对参考基因的表达稳定性进行排序，结果表明 EF-1β和 ACT-2 是两个最稳定的基因，而 GAPDH 和 26S rRNA 不适合这些组织中 qRT-PCR 的归一化。同时还平行确定了角鲨烯合酶编码基因（SS）的表达模式。当 qRT-PCR 数据归一化为 EF-1β和 ACT-2 中的一种或两种表达时，分析结果非常一致，进一步证实了 EF-1β和 ACT-2 作为最佳内参的可靠性。本研究为铁皮石斛转录谱中准确数据归一化提供了重要线索，这对于有价值药用植物的进一步功能基因组学研究至关重要。
