Systematic assessment of commercially available low-input miRNA library preparation kits. Systematic assessment of commercially available low-input miRNA library preparation kits

We compared the performance of six commercial kits (QIAseq, SMARTer, CATS, CleanTag, srLp, TailorMix) capable of handling <100ng total RNA input for miRNA detection sensitivity, reliability, titration response and ability to detect differentially expressed miRNAs at different amounts of synthetic miRNAs. We observed differences in detection sensitivity between the kits, but none were able to detect the full repertoire of expected miRNAs. The reliability within the replicates of all kits was good, while larger differences were observed between the kits, although none could accurately quantify the majority of miRNAs. Overall design: Systematic comparison of miRNA profiles assessed in low-input synthetic miRNA samples and samples from pools of blood derived CD8 T cells of rheumatoid arthritis (RA) patients or healthy controls (HC) by six commercially available library preparation kits. Altogether five different miRNA mixes were created (denoted mix A to mix E). Mix A and Mix B consisted of the equimolar miRNA pool supplemented with the non-equimolar pool present at eight different concentration ratios between the two mixes spanning a 100 fold range. Mix C was a titration of 0.75 mix A and 0.25 mix B, while mix D was a titration of 0.25 mix A and 0.75 mix B. In the case of mixes A-D, the total miRNA concentration was 30 nM, with individual equimolar miRNAs present at 30 pM and other miRNAs ranging from 3-300 pM. Mix E consisted of the same miRNAs as mix A but at a 10-fold lower concentration.

本研究针对6款可处理总RNA输入量<100ng的商用试剂盒（QIAseq、SMARTer、CATS、CleanTag、srLp、TailorMix），比较其在微小核糖核酸(miRNA)检测灵敏度、可靠性、梯度响应能力，以及不同合成miRNA用量下检测差异表达miRNA的性能。研究观察到各试剂盒的检测灵敏度存在差异，但无一能够检测到全部预期的miRNA种类。所有试剂盒的重复样本内部可靠性均表现良好，但不同试剂盒间的差异更为显著；且无一能够准确定量大多数miRNA。整体实验设计：采用6款商用文库制备试剂盒，对低输入量合成miRNA样本，以及类风湿关节炎(RA)患者或健康对照(HC)的血液来源CD8阳性T细胞混合样本中的miRNA表达谱开展系统性比较分析。共制备5种不同的miRNA混合体系（命名为混合液A至混合液E）。混合液A与混合液B由等摩尔miRNA池与不等摩尔miRNA池构成，二者以8种不同浓度比进行配比，浓度跨度达100倍。混合液C为0.75份混合液A与0.25份混合液B的梯度混合体系，混合液D则为0.25份混合液A与0.75份混合液B的梯度混合体系。针对混合液A至D，总miRNA浓度为30 nM，其中单种等摩尔miRNA的浓度为30 pM，其余miRNA的浓度范围为3~300 pM。混合液E与混合液A包含相同的miRNA种类，但浓度降至原水平的1/10。