Differences in microRNA detection levels are technology and sequence dependent
Differences in microRNA detection levels are technology and sequence dependent
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DOI：
10.1261/rna.036475.112
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发表时间：
2013-04-01
期刊：
影响因子：
4.5
通讯作者：
Feldmesser, Ester
中科院分区：
文献类型：
--
作者：
Leshkowitz, Dena;Horn-Saban, Shirley;Feldmesser, Ester
小rna的鉴定和定量具有挑战性，因为它们长度短，在microRNA （miRNA）家族中序列相似性高，并且存在miRNA同种异构体和o -甲基3'修饰。本研究对Agilent和Affymetrix微阵列和Illumina下一代测序这三个高通量商业平台的检测性能进行了系统、全面的比较。研究表明，检测miRNA的能力强烈依赖于平台和miRNA修饰和序列。使用合成转录物，包括成熟的、前体的和o -甲基修饰的mirna插入到人类RNA中，观察到所有插入的mirna的强度变化很大，并且在测试平台之间检测o -甲基修饰mirna的能力降低。此外，还评估了各平台上的内源性人类miRNA表达水平。检测到的miRNA表达水平在不同平台之间不一致。虽然先前描述了miRNA检测中的偏差，但这里以受控的方式在多个平台上研究了这些偏差的终点结果，即检测强度。对包括碱基组成、序列结构和同型miRNA属性在内的大量属性的详细探索表明，它们对miRNA表达检测水平的影响。本研究为理解调整平台相关检测偏差时应考虑的属性提供了基础。
Identification and quantification of small RNAs are challenging because of their short length, high sequence similarities within microRNA ( miRNA) families, and the existence of miRNA isoforms and O-methyl 3' modifications. In this study, the detection performance of three high-throughput commercial platforms, Agilent and Affymetrix microarrays and Illumina next-generation sequencing, was systematically and comprehensively compared. The ability to detect miRNAs was shown to depend strongly on the platform and on miRNA modifications and sequence. Using synthetic transcripts, including mature, precursor, and O-methyl-modified miRNAs spiked into human RNA, a large intensity variation in all spiked-in miRNAs and a reduced capacity in detecting O-methyl-modified miRNAs were observed between the tested platforms. In addition, endogenous human miRNA expression levels were assessed across the platforms. Detected miRNA expression levels were not consistent between platforms. Although biases in miRNA detection were previously described, here the end-point result, i.e., detection intensity, of these biases was investigated on multiple platforms in a controlled fashion. A detailed exploration of a large number of attributes, including base composition, sequence structure, and isoform miRNA attributes, suggests their impact on miRNA expression detection level. This study provides a basis for understanding the attributes that should be considered to adjust platform-dependent detection biases.