总 RNA 纯化试剂盒 - 旋转柱

概述

这些试剂盒适用于从一系列样本中分离总 RNA，包括细胞、细菌、酵母、病毒和体液（包括血浆/血清、血液、唾液、脑脊液等）。提取优质、高纯度的 RNA，其 RIN 值和 A260/A280 均非常出色，适用于 qRT-PCR、RT-PCR、微阵列、NGS 等下游应用。这些试剂盒可在同一馏分中纯化各种大小的 RNA，从大 mRNA、lncRNA 到 microRNA (miRNA)，无需使用苯酚。以相同的速度分离所有 RNA 序列，而不论其大小。此外，当 RNA 序列较小（如 miRNA）时，无论其 GC 含量如何，色谱柱都能与小 RNA 结合。

细节

Supporting Data

Figure 1 / 16

Figure 1. High Quality of Isolated RNA with Complete Size Range.

Unlike most competitors' kits, Norgen's Total RNA Purification Kit allows for the isolation of all sizes of RNA, from the very large RNA down to the microRNA, without the use of phenol. Total RNA was isolated from 1 x 10⁹ *E. coli* cells using Norgen’s Total RNA Purification Kit and a competitor’s kit. Five microliters and 1 µL of the 50 µL isolated RNA was analyzed on an agarose gel (Panel A) and the Agilent® 2100 BioAnalyzer RNA Nano 6000 chip (Panel B), respectively. Note the presence of small RNA species (red square) in the samples isolated via Norgen's kit and the absence of these RNA species in the competitor RNA preparation.

Figure 2. Amplification of Both Large and Small RNAs in the Same Extraction.

Norgen's Total RNA Purification Kit isolates the complete size range of RNA, including small RNAs without the use of phenol. Total RNA was isolated from 0.75 million HeLa cells using Norgen's Total RNA Purification Kit, various silica-based competitors and phenol-based TRI Reagent. RT-PCR was performed according to Shi and Chiang (2005). Fifteen microliters of the 50 µL isolated RNA were polyadenylated in a 50 µL Poly-(A)-Polymerase reaction. Four microliters of the polyadenylated RNA were used in a 20 µL reverse transcription reaction with a poly T adaptor primer. One microliter of the reverse transcription was used in a 20 µL qPCR reaction with primers against the human microRNAs (*miR-19* and *miR-21*) and large mRNA (*S15*). Only the use of Norgen's Total RNA Purification Kit resulted in detection of both microRNAs and mRNA amplification products similar to Competitor 2's kit but without the use of phenol.

Figure 3. High Quality of RNA from a Diverse Range of Inputs.

Norgen's Total RNA Purification Kit allows RNA isolation from a wide range of species or tissue types. Total RNA was isolated from 5 x 10⁸ *E. coli* cells, 1 x 10⁶ HeLa cells, 100 µL rat blood and 10 mg hamster kidney using Norgen's Total RNA Purification Kit. One microliter of the 50 µL isolated RNA was analyzed on the Agilent® 2100 BioAnalyzer using an RNA Nano 6000 chip. Note the integrity of RNA from all inputs with the presence of small RNA species. Norgen's Total RNA Purification Kit consistently isolates high quality RNA from various inputs that score a RIN value between 8 and 10.

Figure 4. Great Isolation Sensitivity.

Norgen's Total RNA Purification Kit allows sensitive RNA extraction from as little as a single cell. Total RNA was extracted from a decreasing number of 293 HEK cells from 1 million cells down to a single cell. Five microliters of the 50 µL isolated RNA was then subjected to a 20 µL reverse transcription using oligo dT primer. Three microliters of the reverse transcription was used in a 20 µL PCR reaction with primers to detect the human beta-actin transcripts. PCR products of beta-actin were detected from as little as a single cell. M is the marker lane.

Figure 5. Linear and Sensitive Isolation of Both Large and Small RNA.

Norgen’s Total RNA Purification Kit allows consistent isolation of both large and small RNA from different input amounts. Total RNA was isolated from 10 to 100,000 HeLa cells using Norgen's Total RNA Purification Kit (blue), a competitor’s silica-based kit (green) and a phenol-based RNA extraction method (red). Relative expression of miR-21 (Panel A), and S15 (Panel B) was determined by RT-qPCR of total RNA samples. In brief, one microliter of the 50 µL isolated RNA was then subjected to a 20 µL reverse transcription using miR-21 stem-loop reverse primer or oligo dT primer. Two microliters of the reverse transcription was used in a 20 µL real-time PCR reaction with primers to detect the human miR-21 (Panel A) and the S15transcripts (Panel B). The resulting threshold cycle (Ct) values were plotted against input cell number. RNA isolated using Norgen's Total RNA Purification had the best linearity (higher R²) and sensitivity (lower Ct) for both large RNA (S15) and small RNA (miR-21).

Figure 6. Effective and Consistent Detection of miRNA from Plasma.

Norgen's Total RNA Purification Kit can effectively isolate miRNA from plasma. Total RNA was isolated from 50, 100 or 200 µL of rat plasma in triplicates using Norgen's Total RNA Purification Kit (blue), a competitor’s silica-based kit (green) and a phenol-based RNA extraction method (red). Stem loop RT-qPCR using primers specific to miR-21 was performed. In brief, two microliters of the 50 µL isolated RNA was then subjected to a 20 µL reverse transcription using miR-21 stem-loop reverse primer or oligo dT primer. Three microliters of the reverse transcription was used in a 20 µL real-time PCR reaction with primers to detect the human miR-21. Norgen's Total RNA Purification Kit is the only product that showed (1) consistent detection of miR-21 transcripts across all input volumes, and (2) Ct values correlated to input volume (decrease Ct with increase input).

Figure 7. Recovering Diverse miRNA Species from Plasma with Better Consistency.

Norgen's Total RNA Purification Kit isolates plasma RNA that performs more consistently in downstream applications such as microarrays. Total RNA including miRNA was isolated from 100 µL of mouse plasma in duplicate using Norgen's Total RNA Purification Kit, Competitor 1's leading miRNA Kit or a phenol-based RNA extraction method. One hundred nanograms of extracted RNA from each kit was applied to an Illumina microRNA expression profiling kit. Scatter plots display better consistency (better clustering) of replicate signals from Norgen's samples. Genes with Pval < 0.01 for both replicates were in blue. The associated table suggested that Norgen's protocol recovered the same diversity of miRNA with better consistency (higher r² value).

Figure 8. Better Diversity of miRNA Detected from Plasma.

Norgen's Total RNA Purification Kit isolates miRNA from plasma with better diversity than a leading competitor. Total RNA including miRNA was isolated from 100 µL of plasma using Norgen's Total RNA Purification Kit or 625 µL of plasma using Competitor A's leading miRNA Kit, and was applied to an NCode expression profiling kit. Microarray images suggested that Norgen's Total RNA Purification Kit (left) isolates a better diversity of miRNA from smaller input amount of plasma than the competitor’s miRNA kit (right). Image courtesy of LC Sciences, Houston.

Figure 9. Better Diversity of miRNA Detected from HeLa Cells using Illumina Small RNA Next Gen Sequencing.

Norgen's Total RNA Purification Kit isolates miRNA from HeLa cells with better diversity than a leading competitor. Total RNA including miRNA was isolated from 1 million HeLa cells using Norgen's Total RNA Purification Kit and Competitor Q's leading miRNA Kit, and was applied to Illumina Small RNA Next Gen Sequencing on a MiSeq sequencer. Panel A showed that Norgen’s Total RNA Purification Kit recovers a higher number of miRNAs than the competitor. In particular, the higher diversity is achieved with a faster and simpler procedure in as little as 20 minutes of RNA sample preparation time without the use of phenol. Panel B is a scatter plot of the average RPM (reads per million) of the miRNAs detected used to compare Norgen and the competitor's kit recovery of miRNA. Norgen's Total RNA Purification Kit recovered a significantly higher number of miRNAs that have higher RPM, as summarized in the graph insert as well as in Panel C.

Figure 10. Consistent Yield of High Quality RNA with Complete Size Range without the Use of Phenol.

Norgen's Total RNA Purification 96-Well Kit allows for the consistent isolation of high quality RNA, with complete sizes ranging from very large RNA down to small RNA without the use of phenol. Total RNA was isolated from 0.5 mL aliquots of an overnight culture of *E. coli* (~5 x 10⁸ cells/mL) using Norgen's Total RNA Purification 96-Well Kit in 16 replicates. Five microliters of the 75 µL isolated RNA were resolved on a 1.2% formaldehyde agarose gel. All 16 replicates showed both high yield and high quality. In addition, all replicates showed effective recovery of all sizes of RNA including the small RNA (arrow).

Figure 13. Great Isolation Sensitivity.

Norgen's Total RNA Purification Kit allows for sensitive RNA extraction from even less than 10 cells. RT-qPCR was used to detect mRNA isolated from various input amounts of HeLa cells, from 100,000 down to a single cell, using Norgen's Total RNA Purification 96-Well Kit. Ten microliters of the 75 µL isolated RNA were then subjected to a 20 µL reverse transcription using oligo dT primers. Three microliters of the reverse transcription were used in a 20 µL real-time PCR reaction with primers to detect the human S15 transcripts. The resulting Ct values were plotted against the input cell number. Total RNA was isolated and detected linearly from as little as a single HeLa cell.

Figure 14. Comparison of total RNA concentration and RNA quality from HeLa cells isolated using column, automated bead-based, and manual bead-based purification methods.

RT-qPCR targeting microRNA-21 showed consistent Ct values (Columns: 21.66; Automation: 19.90; Manual: 19.91), confirming effective small RNA recovery with all three methods

Figure 15. RT-qPCR analysis of microRNA-21 to evaluate small RNA recovery from total RNA purified by different methods.

Total RNA isolated from HeLa cells using manual, automated, and column-based methods was reverse-transcribed and amplified for microRNA-21. All three methods provided comparable Ct values (Automation: 14.89; Columns: 15.48; Manual: 15.57), demonstrating effective and consistent small RNA recovery across platforms.

Figure 16. Total RNA integrity comparison using different purification methods from 1 × 10⁶ HeLa cells.

Total RNA was extracted using the Total RNA Purification Kit (Magnetic Bead System) (Product #75400) on three different platforms: Isopure, KingFisher, and the manual Column Kit method. Each lane was loaded with 5 µL of eluate and run on a 1× MOPS / 10% formaldehyde agarose gel to assess RNA integrity. Distinct 28S and 18S rRNA bands (indicated by arrows) demonstrate high-quality RNA across all platforms. The consistency in band intensity and sharpness suggests efficient RNA recovery and integrity across all methods tested.

支持数据

图 1.分离的 RNA 质量高，大小范围全。与大多数竞争对手的试剂盒不同，Norgen 的总 RNA 纯化试剂盒可以分离所有大小的 RNA，从超大 RNA 到 microRNA，而无需使用苯酚。使用 Norgens 总 RNA 纯化试剂盒和竞争对手试剂盒从 1 x 109 个大肠杆菌细胞中分离出总 RNA。在琼脂糖凝胶（面板 A）和 Agilent® 2100 BioAnalyzer RNA Nano 6000 芯片（面板 B）上分别分析了从 50 µL 中分离出来的 5 µL 和 1 µL 的 RNA。注意通过 Norgen 试剂盒分离的样本中存在小 RNA（红色方框），而竞争对手 RNA 制备中不存在这些 RNA。

图 2.在同一提取物中同时扩增大分子和小分子 RNA。Norgen 的总 RNA 纯化试剂盒无需使用苯酚即可分离出完整大小范围的 RNA，包括小 RNA。使用 Norgen 总 RNA 纯化试剂盒、各种硅胶竞争剂和苯酚 TRI 试剂从 75 万个 HeLa 细胞中分离出总 RNA。RT-PCR 按照 Shi 和 Chiang（2005 年）的方法进行。从 50 µL 中分离出来的 15 µL 的 RNA 在 50 µL Poly-(A)-Polymerase 反应中进行多聚腺苷酸化。在 20 µL 的反转录反应中使用了 4 µL 多聚腺苷酸化的 RNA，并使用了 poly T 适应子引物。在 20 µL q 聚合酶链式反应中使用了 1 µL 的反转录产物，并使用了针对人类 microRNA（miR-19 和 miR-21）和大 mRNA（S15）的引物。只有使用 Norgen 的总 RNA 纯化试剂盒才能检测到 microRNA 和 mRNA 扩增产物，与竞争对手 2 的试剂盒类似，但不使用苯酚。

图 3.从各种输入中获得优质 RNA。Norgen 的总 RNA 纯化试剂盒可从多种物种或组织类型中分离 RNA。使用 Norgen 总 RNA 纯化试剂盒从 5 x 108 个大肠杆菌细胞、1 x 106 个 HeLa 细胞、100 µL 大鼠血液和 10 mg 仓鼠肾脏中分离出总 RNA。在 Agilent® 2100 生物分析仪上使用 RNA Nano 6000 芯片对从 50 µL 中分离出来的 1 µL 的 RNA 进行分析。注意所有输入的 RNA 都是完整的，但存在小 RNA 物种。Norgen 的总 RNA 纯化试剂盒能从 RIN 值在 8 到 10 之间的各种输入物中持续分离出优质 RNA。

图 4.极大的隔离灵敏度。Norgen 的总 RNA 纯化试剂盒可从单个细胞中灵敏提取 RNA。从 293 HEK 细胞（从 100 万个细胞到单个细胞）中提取总 RNA。然后用寡聚 dT 引物对从 50 µL 中分离出来的 5 µL 的 RNA 进行 20 µL 反转录。在 20 µL 聚合酶链式反应中使用了 3 µL 反转录液和引物，以检测人 β 肌动蛋白转录本。只要一个细胞就能检测到 β 肌动蛋白的聚合酶链式反应产物。M 是标记泳道。

图 5.线性、灵敏地分离大分子和小分子 RNA。Norgens 总 RNA 纯化试剂盒可从不同的输入量中稳定地分离出大 RNA 和小 RNA。使用 Norgen 的总 RNA 纯化试剂盒（蓝色）、基于竞争对手硅胶的试剂盒（绿色）和基于苯酚的 RNA 提取方法（红色）从 10 到 100,000 个 HeLa 细胞中分离出总 RNA。通过总 RNA 样本的 RT-qPCR 测定 miR-21（A 组）和 S15（B 组）的相对表达。简言之，从 50 µL 分离的 RNA 中取 1 µL ，使用 miR-21 茎环反向引物或 oligo dT 引物进行 20 µL 反转录。在 20 µL 实时聚合酶链式反应中使用了 2 µL 的反转录产物和引物，以检测人类 miR-21（A 组）和 S15 转录物（B 组）。得出的阈值周期 (Ct) 值与输入细胞数相对应。对于大 RNA (S15) 和小 RNA (miR-21)，使用 Norgen 总 RNA 纯化技术分离的 RNA 具有最佳的线性度（R2 较高）和灵敏度（Ct 较低）。

图 6.从血浆中有效、一致地检测 miRNA。Norgen 的总 RNA 纯化试剂盒能有效地从血浆中分离 miRNA。使用 Norgen 的总 RNA 纯化试剂盒（蓝色）、基于竞争对手硅胶的试剂盒（绿色）和基于苯酚的 RNA 提取方法（红色），从 50、100 或 200 µL 大鼠血浆中分离出总 RNA，每次分离三份。使用 miR-21 的特异性引物进行了干环 RT-q 聚合酶链式反应。简言之，从 50 µL 分离出的 RNA 中取 2 µL ，使用 miR-21 茎环反向引物或 oligo dT 引物进行 20 µL 反转录。在 20 µL 的实时聚合酶链式反应中使用了 3 µL 的反转录产物和引物来检测人类 miR-21。Norgen 的总 RNA 纯化试剂盒是唯一能显示 (1) 在所有输入量下都能一致检测到 miR-21 转录本，以及 (2) Ct 值与输入量相关（输入量增加，Ct 值降低）的产品。

图 7.以更好的一致性从血浆中回收多种 miRNA。Norgen 的总 RNA 纯化试剂盒分离出的血浆 RNA 在微阵列等下游应用中表现更稳定。使用 Norgen 的总 RNA 纯化试剂盒、竞争对手 1 的主要 miRNA 试剂盒或基于苯酚的 RNA 提取方法，从 100 µL 小鼠血浆中分离出包括 miRNA 在内的总 RNA，一式两份。每个试剂盒提取的 100 ng RNA 被用于 Illumina microRNA 表达谱分析试剂盒。散点图显示 Norgen 样本的重复信号具有更好的一致性（更好的聚类）。两次重复中 Pval 均小于 0.01 的基因用蓝色表示。相关表格显示，Norgen 的方案恢复了同样多样性的 miRNA，而且一致性更好（r2值更高）。

图 8.从血浆中检测到的 miRNA 更具多样性。Norgen 的总 RNA 纯化试剂盒能从血浆中分离出 miRNA，其多样性优于主要竞争对手。使用 Norgen 的总 RNA 纯化试剂盒从 100 µL 的血浆中分离出包括 miRNA 的总 RNA，或使用竞争对手 A 的领先 miRNA 试剂盒从 625 µL 的血浆中分离出总 RNA，并将其应用于 NCode 表达谱分析试剂盒。微阵列图像显示，与竞争对手的 miRNA 试剂盒（右图）相比，Norgen 的总 RNA 纯化试剂盒（左图）能从较少的血浆输入量中分离出更多样的 miRNA。图片由休斯顿 LC Sciences 提供。

图 9.利用 Illumina Small RNA 下一代测序技术提高从 HeLa 细胞中检测到的 miRNA 的多样性。Norgen 的总 RNA 纯化试剂盒能从 HeLa 细胞中分离出 miRNA，其多样性优于主要竞争对手。使用 Norgen 的总 RNA 纯化试剂盒和竞争对手 Q 的领先 miRNA 试剂盒从 100 万个 HeLa 细胞中分离出包括 miRNA 在内的总 RNA，并将其应用于 MiSeq 测序仪上的 Illumina Small RNA 下一代测序。面板 A 显示，诺健总 RNA 纯化试剂盒回收的 miRNA 数量高于竞争对手。特别是，在不使用苯酚的情况下，只需 20 分钟的 RNA 样本制备时间，就能以更快、更简单的程序实现更高的多样性。B 组是检测到的 miRNA 平均 RPM（百万读数）散点图，用于比较 Norgen 和竞争对手试剂盒的 miRNA 回收率。Norgens 总 RNA 纯化试剂盒回收的具有较高 RPM 的 miRNA 数量明显较多，如插图和面板 C 所示。

图 10.无需使用苯酚，即可获得稳定的优质 RNA，且大小范围齐全。Norgen 的总 RNA 纯化 96 孔试剂盒可以稳定地分离出优质 RNA，从超大 RNA 到小 RNA，粒度齐全，无需使用苯酚。使用 Norgen 的总 RNA 纯化 96 孔试剂盒，从大肠杆菌（约 5 x108个细胞/mL）过夜培养物的 0.5 mL 等分样本中分离总 RNA，重复 16 次。在 1.2% 甲醛琼脂糖凝胶上从 75 µL 中分离出来的 5 µL 的 RNA。所有 16 个重复均显示出高产和优质。此外，所有重复均显示有效回收了所有大小的 RNA，包括小 RNA（箭头）。

图 11.从各种输入中获得优质 RNA。Norgen 的总 RNA 纯化 96 孔试剂盒可从多种物种和组织类型中分离 RNA。使用 Norgen 总 RNA 纯化 96 孔试剂盒从 8 mg 脑组织（A 泳道）、肾组织（B 泳道）、肝组织（C 泳道）、肺组织（D 泳道）和脾组织（E 泳道）、7.5 x105 HeLa 细胞（F 泳道）和 CHO 细胞（G 泳道）以及 5 x108个细菌（H 泳道）中分离总 RNA。在 1.2% 甲醛琼脂糖凝胶上从 75 µL 中分离出来的 5 µL 的 RNA。通过观察甲醛-琼脂糖凝胶可以看出，Norgen 试剂盒可用于从多种类型的样本中成功分离总 RNA，包括小 RNA 物种。

图 12.大 RNA 和小 RNA 的分离。Norgen 的总 RNA 纯化 96 孔试剂盒可以稳定地分离大 RNA 和小 RNA。使用 Norgen 的总 RNA 纯化 96 孔试剂盒从 5 x105 个 HeLa 细胞样本中分离出总 RNA。然后将每个总 RNA 样本的等分样本用于 2 个不同的 RT-q 聚合酶链式反应。用 miR-21 茎环反向引物或 oligo dT 对从 75 µL 中分离出来的 10 µL 的 RNA 进行 20 µL 反转录。在 20 µL 的实时聚合酶链式反应中使用 3 µL 的反转录物，引物分别用于检测人类 miR-21（A 组）和 S15 转录本（B 组）。所有样本的 mRNA 和 microRNA 扩增结果一致，Ct 值的变异性较低。因此，使用该试剂盒可以持续分离出这两种类型的 RNA。

图 13.极高的隔离灵敏度Norgen 的总 RNA 纯化试剂盒可从少于 10 个细胞中灵敏提取 RNA。使用 Norgen 的总 RNA 纯化 96 孔试剂盒，用 RT-qPCR 检测从不同输入量的 HeLa 细胞（从 100,000 个细胞到单个细胞）中分离出来的 mRNA。然后用寡聚 dT 引物对从 75 µL 中分离出来的 10 µL 的 RNA 进行 20 µL 反转录。在 20 µL 的实时聚合酶链式反应中使用了 3 µL 的反转录物，并使用引物检测人类 S15 转录本。得出的 Ct 值与输入的细胞数相对应。从单个 HeLa 细胞中分离并线性检测总 RNA。

试剂盒规格
最大结合能力	高达 50 μg RNA
最大装载量	650 μL
纯化的 RNA 大小	所有大小，包括小 RNA（< 200 nt）
起始材料的最大用量
动物细胞	3 x 10⁶ 个细胞
动物组织	10 mg （适用于大多数组织*
血液	100 μL
血浆/血清	200 μL
细菌	1 x 10⁹ 个细胞
酵母	1 x 10⁸ 个细胞
真菌	50 mg
植物组织	50 mg
完成 10 次净化所需的时间	20 分钟
平均产量
HeLa 细胞 (1 x 10⁶ 个细胞)	15 μg
大肠杆菌 (1 x 10⁹ 个细胞)	50 μg

* 如需从大量组织中分离总 RNA，请使用 Norgen 的动物组织 RNA 纯化试剂盒（类别号：25700）

储存条件和产品稳定性
所有溶液都应密封保存在室温下。这些试剂盒自发货之日起 2 年内保持稳定。

Documentation

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