从福尔马林固定、石蜡包埋的组织切片中纯化总RNA

  • 创新的方法用于克服福尔马林造成的交联
  • 有效的释放RNA而不破坏其完整性
  • 高效的实验方案,在85分钟内即可获得纯化的RNA

RNeasy FFPE Kit专为从福尔马林固定、石蜡包埋的组织切片中纯化总RNA而设计。独特的裂解和孵育条件逆转福尔马林对RNA的修饰。并且裂解缓冲液可将RNA从组织切片中有效释放出来,防止RNA的进一步降解。试剂盒同样使用DNase和DNase Booster Buffer优化,去除基因组DNA的污染。使用RNeasy MinElute离心柱纯化获得的总RNA,洗脱体积可小至10 μl。整个纯化流程可在QIAcube全自动核酸纯化仪上全自动进行。

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RNeasy FFPE Kit (50)
50 RNeasy MinElute Spin Columns, Collection Tubes, Proteinase K, RNase-Free DNase I, DNase Booster Buffer, RNase-Free Buffers, RNase-Free Water
73504
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The RNeasy FFPE Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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Reliable results in real-time RT-PCR analysis.|Greater yields of RNA.|RNeasy FFPE procedure.|Successful real-time RT-PCR analysis.|Recovery of all usable RNA.|
Total RNA was purified from a 10 µm rat lung FFPE sample using the RNeasy FFPE Kit or alternative kits from Supplier EIII, Supplier N, Supplier AIV, and Supplier M. Real-time RT-PCR assays for PGK1 (phosphoglycerate kinase 1) were performed on the Rotor-Gene Q cycler with (+RT) or without (-RT) the reverse transcription step. The high CT value in the -RT sample from the RNeasy FFPE Kit indicates that purified RNA is virtually free of genomic DNA. Therefore the CT value in the +RT sample from the RNeasy FFPE Kit is a reliable measure of RNA expression.|RNA was purified from 10 µm FFPE sections of the indicated rat tissues using either the RNeasy FFPE Kit or alternative kits from Supplier N, Supplier AIV, and Supplier M. RNA yields were determined by measuring absorbance at 260 nm.|After paraffin is removed from samples, the optimized lysis buffer allows sample lysis with proteinase K digestion in only 15 minutes without sacrificing RNA yields. After lysis, samples are incubated at 80ºC for 15 minutes to reverse formalin crosslinking. Genomic DNA, including small DNA fragments found in FFPE samples, is then effectively removed using DNase and DNase Booster Buffer. Finally, concentrated RNA is purified using RNeasy MinElute spin columns. Since RNA is eluted in a volume of just 14–30 µl, smaller reaction volumes are possible in downstream applications.|Total RNA was purified from rat kidney using the RNeasy FFPE Kit. Real-time RT-PCR assays for PGK1 (phosphoglycerate kinase 1) were performed on the Rotor-Gene Q cycler with (+RT) or without (-RT) reverse transcriptase. The -RT curves demonstrated that RNA purified using the RNeasy FFPE Kit was virtually free of genomic DNA.|RNA purified from 6-month-old FFPE rat liver using the RNeasy FFPE Kit or alternative kits from Supplier AIV, Supplier N, or Supplier M, was analyzed on the Agilent 2100 Bioanalyzer. The peaks indicate that more intact RNA was purified using the RNeasy FFPE Kit than when using alternative kits.|
Performance
使用RNeasy FFPE Kit从固定和包埋的样本中纯化获得的RNA的产量比用其他方法更高(参见"Greater yields of RNA")。回收得到的完整RNA最小可达70 nt,并且几乎没有基因组DNA的污染(参见"Recovery of all usable RNA")。用该试剂盒从FFPE样本中纯化得到的RNA片段非常适用于real-time RT-PCR等敏感的下游应用(参见"Reliable results in real-time RT-PCR analysis"和"Successful real-time RT-PCR analysis")。
Principle

用福尔马林固定组织会导致RNA-RNA和RNA-蛋白质的交联,这会影响RNA在酶学分析中的性能。FFPE样本固定和包埋同样会导致核酸的严重片段化。RNeasy FFPE Kit提供了独特的裂解和孵育条件来逆转福尔马林对RNA的修饰。裂解缓冲液能使RNA从FFPE组织样本中有效释放,并防止进一步的RNA降解。经优化的条件能纯化得到所有可用的RNA,使用RNeasy FFPE Kit从FFPE样本中得到的RNA的产量比其他方法更高。

Procedure

整个RNeasy FFPE操作流程可在85分钟内完成(参见"RNeasy FFPE procedure")。使用蛋白酶K裂解样本仅需15分钟。裂解后,样本在80ºC下孵育15分钟。之后,用DNase处理能有效去除样本中的基因组DNA,包括小的DNA片段。最后,使用RNeasy MinElute离心柱即可收集纯化的RNA,洗脱体积14–30 µl。纯化过程可在QIAcube全自动核酸纯化仪上全自动进行。

Applications

RNeasy FFPE Kit可纯化FFPE 样本中所有大于70 nt RNA,得到适用于RT-PCR等众多下游应用的RNA片段。但是,从FFPE样本中纯化得到的RNA会严重片段化,不能用于需要完整RNA的下游应用。如果使用片段化RNA可能需要做些修饰(例如在RT-PCR时设计小的扩增子)。进行cDNA合成时,不能使用oligo-dT引物,而要使用随机或基因特异性引物。

Feature
Specifications
Applications PCR, qPCR, real-time RT-PCR, microarray
Elution volume 14–30 µl
Format Spin column
Main sample type FFPE tissue samples
Processing Manual (centrifugation), automated (QIAcube)
Purification of total RNA, miRNA, poly A+ mRNA, DNA or protein RNA
Sample amount 1*5 µm to 4*10µm sections
Technology Silica technology
Time per run or per prep 70 minutes
Yield Varies

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Kit Handbooks
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For purification of total RNA from formalin-fixed, paraffin-embedded tissue sections
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Safety Data Sheets
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