生物體表現(xiàn)出的各種特性,主要是由基因的差異表達引起的,這種表達的變化是調控細胞生命活動過程的核心機制,通過比較同一類細胞在不同生理條件下或在不同生長發(fā)育階段的基因表達差異,可為分析生命活動過程提供重要信息。在研究WP3在不同鹽濃度,溫度中的基因差異表達時,它的全基因組測序工作還正在進行之中,當時在對WP3基因組序列了解不多的情況下選用了原核生物特異的差異顯示研究方法——RNA隨機引物PCR(RAP_PCR)[6_7]。與文獻報道一樣[8],本實驗結果顯示,測序膠電泳后出現(xiàn)差別條帶太多,假陽性率高達70%左右,重復性差,且對高豐度的RNA具很強的傾向性。在有差異的顯示片段中難以知道哪些基因是已知的,哪些是未知的。本次實驗得到的有差異的擴增條帶短(110~450bp)。如何有效的從備選的差異片段中進一步驗證真正差異表達的基因是很重要的。作者分別用3種方法驗證了從變性長膠聚丙烯酰胺凝膠電泳得到的片段,結果表明,RT_PCR靈敏性最強,RNA點雜交需要的RNA量最大,熒光實時定量PCR精確性最高。因此,在實驗條件允許下,用熒光實時定量PCR對較少片段的驗證較好,此法需要的RNA樣品少,基因差異表達的變化可以量化。
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