Effect of long-term storage in TRIzol on microarray-based gene expression profiling.
Effect of long-term storage in TRIzol on microarray-based gene expression profiling.
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DOI：
10.1158/1055-9965.epi-10-0565
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发表时间：
2010-10
期刊：
影响因子：
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通讯作者：
Davis RE
中科院分区：
文献类型：
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作者：
Ma W;Wang M;Wang ZQ;Sun L;Graber D;Matthews J;Champlin R;Yi Q;Orlowski RZ;Kwak LW;Weber DM;Thomas SK;Shah J;Kornblau S;Davis RE
尽管TRIzol被广泛用于RNA的保存和分离，但人们怀疑，在TRIzol中长时间保存样品可能会影响基于阵列的基因表达谱（GEP），通过在逆转录（RT）过程中过早终止。Illumina阵列上的GEP比较了多发性骨髓瘤（MM）和急性髓性白血病（AML）原发样品的配对同种异体（冷冻保存或TRIzol保存）。数据在“探针水平”（单一共识值）或“头水平”（由单个珠提供的多次测量）进行分析。TRIzol储存不会影响样品组之间的标准探针水平比较：不同的保存方法不会在MM或AML样品组中产生差异表达探针（dep），也不会实质性地影响区分这些组的许多dep。通过基因集富集分析发现了差异，但由于样品标记排列不稳定、TRIzol等分限制不平衡、MM和AML组之间不一致以及缺乏生物学合理性，这些差异被忽略。珠级比较发现样本对中有许多dep，但大多数（73%）变化<2倍。没有一致的证据表明TRIzol导致RT过早终止。相反，在TRIzol保存的样品中，来自转录本5 '端附近探针的信号增加，系统地引起了dep的一个子集，这表明TRIzol可以更好地保存mRNA。TRIzol很好地保留了RNA质量，对GEP没有有害影响。用TRIzol和不加TRIzol冷冻的样品可以用GEP进行比较，对系统伪像的影响很小。在TRIzol中延长样品储存的标准做法适用于GEP。
Although TRIzol is widely used for preservation and isolation of RNA, there is suspicion that prolonged sample storage in TRIzol may affect array-based gene expression profiling (GEP), via premature termination during reverse transcription (RT). GEP on Illumina arrays compared paired aliquots (cryopreserved or stored in TRIzol) of primary samples of multiple myeloma (MM) and acute myeloid leukemia (AML). Data were analyzed at the “probe level” (a single consensus value) or “bead level” (multiple measurements provided by individual beads). TRIzol storage does not affect standard probe-level comparisons between sample groups: different preservation methods did not generate differentially-expressed probes (DEPs) within MM or AML sample groups, or substantially affect the many DEPs distinguishing between these groups. Differences were found by gene set enrichment analysis, but were dismissible because of instability with permutation of sample labels, unbalanced restriction to TRIzol aliquots, inconsistency between MM and AML groups, and lack of biological plausibility. Bead-level comparisons found many DEPs within sample pairs, but most (73%) were <2-fold changed. There was no consistent evidence that TRIzol causes premature RT termination. Instead, a subset of DEPs were systematically due to increased signals in TRIzol-preserved samples from probes near the 5’ end of transcripts, suggesting better mRNA preservation with TRIzol. TRIzol preserves RNA quality well, without a deleterious effect on GEP. Samples stored frozen with and without TRIzol may be compared by GEP with only minor concern for systematic artifacts. The standard practice of prolonged sample storage in TRIzol is suitable for GEP.