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查看更多产品信息 RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE - FAQs (AM1975)
10 个常见问题解答
对于福尔马林固定样本，我们推荐使用RecoverAllFFPE样品总核酸分离试剂盒。您可以使用此试剂盒从FFPE样品中提取总RNA和miRNA，然后按照miRVana试剂盒说明中的富集方案对上述样本内的miRNA进行富集。对于未固定的LCM样品，您可以使用RNAqueous试剂盒。
许多因素都可能影响从FFPE组织提取的DNA的总体质量和得率。以下为我们就如何解决这些关键影响因素而提出的一些建议：
1.组织获取和样本制备 - 在条件允许的情况下，组织应在外科切除下来后一小时内固定。使用中性福尔马林或多聚甲醛缓冲液固定的最佳时长为12-24小时。固定后的组织进入包埋程序之前，应彻底脱水。
2.组织块的储存 -在条件允许的情况下，储存的组织块尽量保证无切面。如果出现切面，氧气、水、光、以及外源感染（如真菌、昆虫等）等其他环境因素可能会造成组织块的进一步破坏。
3.用于DNA分离的组织类型、大小和数量 – 建议的组织厚度为10-20 µm。所用的切片数量取决于组织类型（决定细胞密度）和表面积大小(推荐大小：50-300 mm2）。过量的起始材料可能导致过滤柱堵塞，导致得率下降。
4.包埋组织时的过量石蜡 - 在条件允许的情况下，在纯化操作之前脱去组织样本上过多的石蜡。对于二甲苯纯化法而言，室温条件下的两次二甲苯处理通常能够彻底脱石蜡。必要情况下，可在更为强效的37–55°C条件下处理达30分钟。在二甲苯脱蜡操作完成后，须对沉淀物进行两次100%乙醇漂洗，再彻底去除100%乙醇。磁珠法采用全新的化学方法来处理石蜡，所选用的组织切片应在20 µm以内。
请点击此处（https://www.thermofisher.com/us/en/home/references/ambion-tech-support/rna-isolation/general-articles/extraction-of-nucleic-acids-from-ffpe-samples.html）阅读更多关于FFPE样本核酸提取的信息。
我们为此提供了两种试剂盒：用于FFPE组织的RecoverAll总核酸分离试剂盒和MagMAX FFPE DNA/RNA Ultra试剂盒。请点击此处（http://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/dna-extraction/genomic-dna-extraction/dna-extractions-working-with-ffpe-samples.html）了解更多关于这些试剂盒的差异。
For recovery of miRNA of formalin-fixed samples, we recommend using RecoverAll Total Nucleic Acid Isolation Kit for FFPE. You can isolate total and miRNA using the RecoverAll Total Nucleic Acid Isolation Kit for FFPE, then use that prep for enrichment of miRNA using the enrichment protocol described in the instructions for the mirVana kit. For unfixed LCM samples, you could use an RNAqueous kit.
There are a number of factors that can impact the overall quality and yield of DNA isolated from FFPE tissues. Here are recommendations to address several key factors:
- Upstream tissue procurement and tissue specimen preparation - if possible, tissues should be fixed within one hour of surgical resection. The optimal fixation time is 12-24 hours using neutral-buffered formalin or paraformaldehyde. Fixed tissues should be thoroughly dehydrated prior to the embedding process.
- Block storage - storage of blocks without cut faces, when possible, prevents ongoing damage from exposure to atmospheric oxygen, water, and other environmental factors such as light and infestation (fungi, insects, etc.).
- Tissue type, size, and amount being used for DNA isolation - the recommended tissue thickness is 10-20 µm. The number of sections used is determined by the tissue type (which impacts cell density) and surface area (recommended size: 50-300 mm^2). Excess starting material can cause filter clogging, resulting in poor yield.
- Excessive amount of paraffin used for embedding tissues - when possible, excess paraffin should be trimmed away prior to starting the purification protocol. For xylene-based purification methods, two xylene treatments at room temperature should be sufficient for complete deparaffinization. If desired, a more rigorous 37-55 degrees C treatment can be performed for up to 30 minutes. After the xylene deparaffinization, it is crucial that the 100% ethanol is completely removed and the pellets are dry after the two 100% ethanol washes. The magnetic bead method employs novel chemistries to deal with the paraffin that limits input to 20 µm sections.
Read more about extraction of nucleic acids from FFPE samples here (http://www.thermofisher.com/us/en/home/references/Invitrogen-tech-support/rna-isolation/general-articles/extraction-of-nucleic-acids-from-ffpe-samples.html).
We offer 2 kits: RecoverAll Total Nucleic Acid Isolation Kit for FFPE and MagMAX FFPE DNA/RNA Ultra Kit Read more about the differences between these kits here (http://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/dna-extraction/genomic-dna-extraction/dna-extractions-working-with-ffpe-samples.html).
We recommend the RecoverAll Total Nucleic Acid Isolation Kit for FFPE (Cat. No. AM1975). This kit is optimized for isolation of both DNA and RNA from formalin or paraformalin-fixed, paraffin-embedded (FFPE).
Another option is TRIzol Reagent, but be sure to check the references listed below. Because paraffin is not soluble in TRIzol Reagent, paraffin-embedded tissues can be quick-heated to get the tissue out of the paraffin; any paraffin which remains will float to the top of the aqueous phase (and should be avoided). (If the slice is very thin, the whole slice can be added to the TRIzol Reagent, and hopefully, the tissue will be exposed to the reagent). Most of the references we surveyed do not provide quantitative data, because paraffin-embedded tissues are dramatically influenced by the action of nucleases prior to fixation and by the formalin fixation time.
The ability to detect specific housekeeping genes by PCR analysis with RNA or DNA extracted from these tissues is usually considered to be a positive result. We do not have a protocol per se, but we have spoken with customers who are doing this. We recommend deparaffinizing with xylene (or other organic), then grinding the sample very thoroughly in TRIzol Reagent (may require a Polytron); in most cases, you have to homogenize with vigor because the DNA is crosslinked and you have to get it free. Microcarrier is recommended since the RNA is crosslinked and fragmented. From this point, the standard isolation protocol can be used. They have found publications that show that the success of the isolation is dependent on how long the sample was fixed (there is an inverse relationship): Inoue, T., et. al., Pathology International (1996) Vol 46, Iss 12, pp. 997-1004.
The main difference between all RNases is where they cleave the RNA (what site they recognize) and whether it is single stranded or double stranded. RNase H is an endoribonuclease that specifically hydrolyzes the phosphodiester bonds of RNA in RNA:DNA duplexes to generate products with 3' hydroxyl and 5' phosphate ends. It will not degrade single-stranded or double-stranded DNA or RNA.
RNase A is an endoribonuclease that specifically hydrolyzes RNA after C and U residues. Cleavage occurs between the 3'-phosphate group of a pyrimidine ribonucleotide and the 5'-hydroxyl of the adjacent nucleotide. The reaction generates a 2':3' cyclic phosphate which then is hydrolyzed to the corresponding 3' nucleoside phosphates.
RNase B is a glycoprotein that possesses an amino acid composition indistinguishable from that of RNase A and contains carbohydrate (6 residues of mannose and 2 residues of N-acetylglucosamine per molecule). It is consequently considered to be a carbohydrate derivative of RNase A. (Reference: Tarentino A et al (1970) J Biol Chem 245:4150.) RNase B has the same specificity as RNase A. (Reference: Plummer T (1963) J Biol Chem 238:1396.)
RNaseOUT RNase inhibitor inhibits RNase A, B, and C but does not inhibit RNase 1, RNase T1, S1 Nuclease, RNase H, RNase T2. Any RNaseOUT RNase inhibitor present from the first-strand synthesis will not cause a problem for the RNase H that is used in second-strand synthesis. RNaseOUT RNase inhibitor will not inhibit DNase I.
For recovery of miRNA from formalin-fixed samples, we recommend using RecoverAll Total Nucleic Acid Isolation Kit for FFPE. You can isolate total and miRNA using the RecoverAll Total Nucleic Acid Isolation Kit for FFPE, then use that prep for enrichment of miRNA using the enrichment protocol described in the instructions for the mirVana kit. For unfixed LCM samples, you could use an RNAqueous kit.
We recommend the following DNA isolation kits:
- RecoverAll Total Nucleic Acid Isolation Kit for FFPE (Cat. No. AM1975)
- Ion Ampliseq Direct FFPE DNA Kit (Cat. Nos. A31133, A31136)
- MagMAX FFPE DNA/RNA Ultra Kit (Cat. No. A31881)
- PureLink Genomic DNA Mini Kit (Cat. Nos, K182000, K182001, K182002)