An optimised direct lysis method for gene expression studies on low cell numbers.
An optimised direct lysis method for gene expression studies on low cell numbers.
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DOI：
10.1038/srep12859
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发表时间：
2015-08-05
影响因子：
4.6
通讯作者：
Dobrovic A
中科院分区：
文献类型：
--
作者：
Le AV;Huang D;Blick T;Thompson EW;Dobrovic A
对单个细胞或有限数量细胞的基因表达分析越来越感兴趣。一种这样的应用是分析收获的循环肿瘤细胞（CTC），其通常以非常低的数量存在。一个高效的RNA提取方案，其中涉及最少的步骤，以避免RNA损失，是必不可少的低输入细胞数。我们比较了几种裂解液，使逆转录（RT）直接对细胞裂解液进行，提供了一种简单快速的方法，以尽量减少RNA损失的RT。裂解液进行了评估，通过逆转录定量聚合酶链反应（RT-qPCR）在低细胞数从四个乳腺癌细胞系分离。我们发现，含有非离子去污剂（IGEPAL CA-630，化学上等同于Nonidet P-40或NP-40）和牛血清白蛋白（BSA）的裂解溶液产生最佳的RT-qPCR产率。这种直接裂解逆转录方案优于使用商业试剂盒的基于柱的提取方法。这项研究证明了一种简单，可靠，时间和成本效益的方法，可广泛用于任何需要从低到非常低的细胞数量制备RNA的情况。
There is increasing interest in gene expression analysis of either single cells or limited numbers of cells. One such application is the analysis of harvested circulating tumour cells (CTCs), which are often present in very low numbers. A highly efficient protocol for RNA extraction, which involves a minimal number of steps to avoid RNA loss, is essential for low input cell numbers. We compared several lysis solutions that enable reverse transcription (RT) to be performed directly on the cell lysate, offering a simple rapid approach to minimise RNA loss for RT. The lysis solutions were assessed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) in low cell numbers isolated from four breast cancer cell lines. We found that a lysis solution containing both the non-ionic detergent (IGEPAL CA-630, chemically equivalent to Nonidet P-40 or NP-40) and bovine serum albumin (BSA) gave the best RT-qPCR yield. This direct lysis to reverse transcription protocol outperformed a column-based extraction method using a commercial kit. This study demonstrates a simple, reliable, time- and cost-effective method that can be widely used in any situation where RNA needs to be prepared from low to very low cell numbers.