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Small RNA建库试剂盒 NEXTflex® Small RNA Sequencing Kit v3


随机性接头,减少小RNA建库中的偏差,并可免除小RNA建库中的凝胶纯化


NEXTflex™ Small RNA Sequencing Kit v3试剂盒使用专利为small RNA建库提供一个可以减少偏差的实验方案,可免除凝胶纯化或使用低样本起始量,这个方案适用于Illumina平台。 Bioo Scientific用以减少连接偏差的方法是在连接步骤中使用随机性接头,相比传统的方法而言,大大减少了偏差的产生。数据中偏差的减少,更加准确的反应出起始材料中small RNA的丰度。此外,还能够在更少的总reads数中检测到更多的miRNAs,提高small RNA的测序效率,降低测序成本。


NEXTflex Small RNA-Seq Kit v3可免除Small RNA建库中的凝胶纯化过程,且降低样本起始量。试剂盒采用两种方法大幅降低接头二聚体的形成,以免除凝胶纯化。样品起始量为≥200 ng的总RNA时,small RNA建库可通过减少接头二聚体的方式完全免除凝胶纯化过程。使用NEXTflex Small RNA-Seq kit v3试剂盒进行免凝胶电泳建库可获得高miRNAs读取率。文库制备的样本起始量可少至1 ng总RNA,需增加PCR循环数,获得无接头二聚体的文库(低样本起始量文库构建通常需要进行凝胶片段选择)


货号名称规格
NOVA-5132-05NEXTflex® Small RNA-Seq Kit v3 (8 barcodes)8 rxns
NOVA-5132-06NEXTflex® Small RNA-Seq Kit v3 (48 barcodes)48 rxns
说明书

产品组分

实验流程

实验数据

引用文献

相关产品


试剂盒组分

NEXTflex™ 3’ 4N Adenylated Adapter
NEXTflex™ 3’ Ligation Buffer
NEXTflex™ 3’ Ligation Enzyme Mix
NEXTflex™ Adapter Depletion Solution
NEXTflex™ Adapter Inactivation Buffer
NEXTflex™ Adapter Inactivation Enzyme
NEXTflex™ 5’ 4N Adapter
NEXTflex™ 5’ Ligation Buffer
NEXTflex™ 5’ Ligation Enzyme Mix
M-MuLV Reverse Transcriptase
NEXTflex™ RT Buffer
NEXTflex™ Universal Primer
NEXTflex™ Barcode Primer
NEXTflex™ Small RNA PCR Master Mix
6X Loading Dye
Ready to Load Low MW Ladder
Resuspension Buffer
Nuclease-free Water
microRNA Control
NEXTflex™ Cleanup Beads
NEXTflex™ Elution Buffer


用户自备材料

1 ng - 2 μg total RNA or purified small RNA from 1-10 μg total RNA in up to 10.5 μL Nuclease-free Water
Isopropanol
80% Ethanol
2, 10, 20, 200 and 1000 μL pipettes
RNase-free pipette tips
Microcentrifuge
96 well PCR Plate Non-skirted (Phenix Research®, Cat # MPS-499) or similar
Thin-wall nuclease-free PCR tubes
Thermocycler
Heat block
Vortex
Magnetic Stand -96 (Thermo Fisher Scientific®, Cat # AM10027) or similar


NEXTflex™ Small RNA-Seq Library Prep Kit v3实验流程.png


4 NEXTflex™ Small RNA Sequencing Kit v3-1.png

使用人类脑部总RNA两次重复建立small RNA文库,于Illumina Miseq平台测序。横坐标表示从不同文库中收集到的reads数目,纵坐标为检测到的iRNA组的平均数,测序数据中一组miRNA有≥20 reads数才定义为被检测到。组成曲线的点为检测到100组iRNA所需要的总reads数量。


低起始量的Small RNA建库方案

减少接头二聚体的技术应用于small RNA建库试剂盒,也可降低文库制备时样本起始量。文库制备的样本起始量可少至5 ng总RNA,增加PCR循环数,获得无接头二聚体的文库。


NEXTflex Small RNA-Seq Kit V3-2.png

Fig.2 显示的是不同样品起始量时表达值的重复性


使用NEXTflex Small RNA-Seq kit v3建库试剂盒,利用起始量为100 ng和10 ng人脑部总RNA样本建库,并分析检测到的miRNA表达的差异。皮尔森相关系数如上显示。


Small RNA-Seq自动化系统匹配

NEXTflex Small RNA-Seq Kit V3建库试剂盒可匹配 PerkinElmer® Sciclone® NGS/NGSx Workstation


NEXTflex与 Competitor's Small RNA-Seq 建库试剂盒比较


1 NEXTflex™ Small RNA Sequencing Kit v3.png

利用起始量为1 ng的Miltenyi miRXplore Universal References(963种miRNAs等摩尔量混合)构建文库并测序,下图为三次重复结果:

4 NEXTflex™ Small RNA Sequencing Kit v3.png


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