For purification of total RNA from formalin-fixed, paraffin-embedded tissue sections
  • Novel method to overcome formalin crosslinking
  • Efficient release of RNA without compromising integrity
  • Streamlined protocol providing RNA in just 85 minutes

The RNeasy FFPE Kit is specially designed for purifying total RNA from formalin-fixed, paraffin-embedded tissue sections. Special lysis and incubation conditions reverse formaldehye modification of RNA. In addition, the lysis buffer efficiently releases RNA from tissue sections while avoiding further RNA degradation. The kit also uses DNase and DNase Booster Buffer for optimized removal of genomic DNA contamination. RNeasy MinElute spin columns enable purification of total RNA with elution volumes of as low as 10 μl. Purification can be fully automated on the QIAcube Connect.


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产品 货号 目录价:
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
询价

RNeasy FFPE Kit适用于分子生物学应用。该产品不适用于疾病的诊断、预防或治疗。


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real-time RT-PCR分析的可靠结果。|RNA产量更高。|RNeasy FFPE实验流程。|成功的real-time RT-PCR分析。|回收各种有用RNA。|
使用RNeasy FFPE Kit或者Supplier EIII、Supplier N、Supplier AIV和Supplier M的同类试剂盒,从10 µm大鼠肺FFPE样本中纯化总RNA。在Rotor-Gene Q实时荧光定量PCR分析仪上进行PGK1(磷酸甘油酸激酶1)的real-time RT-PCR分析,包括(+RT)或不包括(-RT)逆转录步骤。使用RNeasy FFPE Kit在-RT样本中获得高CT值,表示纯化的RNA中几乎不含基因组DNA。因此,使用RNeasy FFPE Kit在+RT样本中获得的CT值代表了可靠的RNA表达水平。|使用RNeasy FFPE Kit或者Supplier N、Supplier AIV和Supplier M的同类试剂盒,从标示的10 µm大鼠肺组织的FFPE切片中纯化RNA。测量260 nm处的吸光值,确定RNA的产量。|从样本中去除石蜡后,在优化的裂解缓冲液中,采用蛋白酶K酶切,只需15分钟即可完成样本裂解,且不影响RNA的产量。裂解后,将样本置于80ºC孵化15分钟,逆转福尔马林交联。然后使用DNase和DNase Booster Buffer高效去除基因组DNA,包括FFPE样本中的小DNA片段。最后,使用RNeasy MinElute离心柱纯化浓缩的RNA。RNA的洗脱体积仅为14–30 µl,因此可以减少下游操作中的反应体积。|使用RNeasy FFPE Kit从大鼠肾脏中纯化总RNA。在Rotor-Gene Q实时荧光定量PCR分析仪上,使用(+RT)或不使用(-RT)逆转录酶进行PGK1(磷酸甘油酸激酶1)的real-time RT-PCR分析。-RT曲线证明,使用RNeasy FFPE Kit纯化的RNA中几乎不含基因组DNA。|在Agilent 2100 Bioanalyzer上使用RNeasy FFPE Kit或者Supplier AIV、Supplier N或Supplier M的同类试剂盒,从6个月的FFPE大鼠肝脏中纯化RNA。峰值表示,与使用其他试剂盒相比,使用RNeasy FFPE Kit可以纯化获得更完整的RNA。|
性能
With the RNeasy FFPE Kit, yields of RNA purified from fixed and embedded samples are greater than those achieved using other methods (see figure “Greater yields of RNA”). Recovered RNA is intact down to 70 nucleotides in length and virtually free of genomic DNA contamination (see figure “Recovery of all usable RNA”). Usable RNA fragments purified with the kit from FFPE samples are highly suited for use in sensitive downstream applications, including real-time RT-PCR (see figures “Reliable results in real-time RT-PCR analysis” and “Successful real-time RT-PCR analysis”).
原理

Fixing tissues with formalin leads to RNA–RNA and RNA–protein crosslinking which impairs RNA performance in enzymatic assays. Fixation and embedding conditions can also result in heavily fragmented nucleic acids in FFPE samples. The RNeasy FFPE Kit uses special lysis and incubation conditions to reverse formaldehyde modification of RNA. The lysis buffer efficiently releases RNA from FFPE tissue samples, preventing further RNA degradation. These optimized conditions allow purification of all usable RNA, leading to greater yields from FFPE samples with the RNeasy FFPE Kit than with other methods.

操作流程
The entire RNeasy FFPE procedure can be completed in as little as 85 minutes (see flowchart "RNeasy FFPE procedure"). Sample lysis with proteinase K digestion requires only 15 minutes. After lysis, samples are incubated at 80ºC for 15 minutes. DNase treatment then effectively removes genomic DNA, including small DNA fragments. Finally, concentrated RNA is purified using RNeasy MinElute spin columns, and eluted in a volume of 14–30 µl. Purification can be fully automated on the QIAcube.
应用

The RNeasy FFPE Kit isolates all RNA molecules longer than 70 nucleotides from FFPE samples, providing usable RNA fragments for numerous downstream applications, including RT-PCR. However, RNA purified from FFPE samples is heavily fragmented and should not be used in downstream applications that require full-length RNA. Some applications may require modifications to allow the use of fragmented RNA (e.g., designing small amplicons for RT-PCR). For cDNA synthesis, either random or gene-specific primers should be used instead of oligo-dT primers.

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For purification of total RNA from formalin-fixed, paraffin-embedded tissue sections
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