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NEB 始终致力于推动生命科学领域的创新与发展。NEB 全新推出的 NEBNext® 低偏差 Small RNA 文库制备试剂盒正是我们这一理念的结晶。它专为制备能准确反映样本中各类 RNA 相对比例的高保真文库而设计，最.大程度减少文库中 Small RNA 种类的偏差，更精准地反映样本中独特 Small RNA 的数量与比例。
技术优势，卓越性能
行业最低偏差：全面分析所有 RNA 种类，避免文库制备过程引入偏差，精准检测miRNA、tRNA 片段、snoRNA、piRNA 等各类小 RNA。
超低起始量：总 RNA 1,000 ng - 0.5 ng；富集后 Small RNA 5 ng - 0.05 ng，获得更可靠的结果。
免凝胶操作：两步磁珠纯化替代传统凝胶回收，3.5 小时完成建库，节省时间。
支持多重测序：最多可搭配 480 种独特双端索引引物（需单独购买）。
18 个月有效保质期：便于实验规划。
科研验证，数据说话
NEBNext® 低偏差 Small RNA 文库制备试剂盒制备的文库偏差性最低
使用 100 种合成对照 miRNA（包含 5 种具有 3' 端 2'-O-甲基化的 miRNA）混合物制备 NEBNext 低偏差 Small RNA 文库。以 0.3 ng 合成 miRNA 混合物为起始样本，比较 NEBNext 低偏差 Small RNA 文库制备试剂盒与 Revvity®（NEXTFLEX® Small RNA Sequencing Kit V4）、Qiagen®（QIAseq® miRNA Library Kit)）和 Illumina（TruSeq® Small RNA Library Prep）制备的文库之间的偏差。文库在 Illumina NextSeq® 500 平台（1 × 56 bp）进行测序，每个文库取 500 万条 reads 进行接头修剪（Flexbar）并使用 STAR (v2.7.8a) 比对至合成序列。预期 reads 值通过比对至对照序列的总 reads 数除以对照序列总数计算得出，图中黑线表示 100% 预期值。计算每个对照序列的观测 reads 与预期 reads 的百分比，并在重复实验间绘图。NEBNext 文库中 90% 的对照 miRNA 位于预期的 2 倍范围内，而竞品仅有 19-30% 在此范围内。竞品试剂盒存在更多低表达序列（50-65%）而非高表达序列（15-24%）。NEBNext 低偏差 Small RNA 文库制备试剂盒制备的文库偏差性始终最低。
The NEBNext Low-bias Small RNA Library Prep Kit minimizes biased representation of small RNA species, more accurately reflecting the number and proportion of unique small RNAs present. NEB's next-generation kit does this via a randomized splint ligation-based approach (see below), which captures small RNAs (<120 nt) with a 5' phosphate and 3' hydroxyl group. Species identified include microRNAs (miRNAs) from a variety of samples (e.g., human, plant), transfer RNAs (tRNAs) and tRNA-derived fragments, small nucleolar RNAs (snoRNAs), and piwi-interacting RNAs (piRNAs). This kit is designed for the preparation of Illumina®-compatible small RNA sequencing libraries.
Benefits:
- Analyze all RNA species present, without library prep-generated bias
- Simplify and streamline your library prep workflow (~3.5 hours), saving time
- Derive insights from biologically relevant input amounts
- Plan ahead with a 18-month shelf life
- Learn a single protocol for standard and 2'-O-methylated samples
- Expand your insights with up to 480 compatible UDI primer pairs, available separately
The NEBNext Low-bias Small RNA Library Prep Kit was developed following the 2020 publication of original research work by NEB scientists in Nucleic Acids Research. The article, "A low-bias and sensitive small RNA library preparation method using randomized splint ligation" was authored by Sean Maguire, Greg Lohman, and Shengzi Guan. Several protocol enhancements have improved the kit to outperform the originally published methods.
This kit is compatible with NEBNext LV Unique Dual Index Primer Pairs Sets 1-5; see neb.com/oligos for more details.
Each kit component must pass rigorous quality control standards and, for each new lot, the entire set of reagents is functionally validated together by construction of an indexed library and sequencing on an Illumina sequencer.
Figure 1: NEBNext Low-bias Small RNA Library Prep workflow
Library construction with the NEBNext Low-bias Small RNA Library Prep Kit is robust and streamlined and can be completed in less than 4 hours. This enhanced workflow features protocol optimizations that enable shorter reaction times with two bead-purification steps that enable increased throughput and ease of use.
Figure 2: NEBNext Low-bias Small RNA Library Prep method
The NEBNext Low-bias Small RNA Library Prep Kit requires 1,000–0.5 ng of total RNA, or 5–0.05 ng of enriched small RNA. For small RNAs to be compatible with the kit, they must have 5´-monophosphate and 3´-hydroxyl ends to enable ligation to Illumina®-compatible adaptors. The workflow features a reagent-additive approach that requires only two bead purification steps. Initially, a 3´ adaptor is ligated to the RNA, and then excess adaptor molecules are removed. Next, two reactions happen simultaneously: the 5´ adaptor is ligated, and the previously ligated 3´ adaptor is enzymatically modified to create the reverse transcription primer. cDNA is then generated, and a bead-based size selection ensures removal of non-ligated adaptor molecules and larger cDNA molecules. PCR amplification makes full-length Illumina-sequenceable molecules and introduces i5 and i7 indices. There are two bead purification strategies that can be used after library amplification. One approach uses a bead size selection that results in overall enrichment of miRNAs. The other method uses bead purification, which results in a generalized small RNA library prep. The NEBNext Low-bias Small RNA Libraries are compatible with NEBNext LV Unique Dual Index Primer Pairs (Sets 1-5)..
