DNA and RNA purification of FFPE tissue samples.

Formarin fixed after paraffin encased intissue referred to simply as FFPE samples.

will be organized in 4-10% of the Formarin quickly fixed. Fixed time limits of 14-24 hours (the longer the fixed time, the easier it will be to fragment<1> <6> to the detriment of downstream experiments). The fixed tissue is completely dehydrated (completely removes the remnants of Formarin because it hinderstheK).

QIAamp DNA FFPE Tissue FFPE tissue sample DNA operation process

remove paraffin: dissolved in xylene and removed

K Incubation

heating 90 degrees C: reverse crosslinking

. . . binding to the upper column: suitable solution environment, DNA binding to silicone film column

. . washing residual contaminants

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. . . . . . . . . . . . . < a href> "> reagent box solution formulation and content New Elution Buffer ATE (regulated to a higher pH, with PCR enzyme more suitable

FFPE sample RNA and miRNA purification principles briefly:

tissue fixed by Folmarin causes RNA-RNA and RNA-protein crosslinking, which affects the performance of RNA in downstream applications. MiRNeasy FFPE Kit provides unique lysate and incubation conditions to reverse the modification of DNA caused by Formarin. This ensures that the resulting RNA is optimally performed in a range of downstream applications, including real-time " The unique lysing buffer night effectively releases RNA from tissue slices while avoiding further explanation of RNA. The kit also applies gDNA removal columns to effectively remove < a href"" > genegroup. Optimised binding conditions ensure the purification of approximately 18 nt or more of RNA. RNA production and performance are superior to other miRNA purification methods, such as phenol-chloroform pumping.

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,221); The optimized lysation buffer night ensures that protease K lysing samples are applied within 15 minutes without affecting RNA yield. After cleavage, incubate the sample at 80oC for 15 minutes, reversing the Formarin crosslink. The gDNA removal column is then applied to quickly remove genomic DNA, and RNeasy MinElute spin columns is applied to purify the concentrated RNA. Since RNA is only washed out to 14-30 μl, the reaction volume in downstream applications can be smaller.

's various literatures summarize the experience of RNA purification:

1, after obtaining the sample, as soon as possible to start fixing.

2, thinn the sample and soak it (preferably if you can reach 5mm or thinner).

3, avoid excessive fixation, soak for too long.

4, RNA purification should include steps to reverse the glue. The steps in QIAGEN's kit are meeting this recommendation.

5, purified samples are preferably fixed or buried within six months of the sample. The longer the placement time, the less conducive it is to RNA purification.

use random quotations or mixtures in subsequent reverse transcription reactions instead of just oligo-dT. When designing reverse transcription PCRs, be careful not to oversleed the amplifier's fragment length.

Figure 1. Recovery of small RNA fragments from FFPE tissues. Agilent bioanalyzer analysiss was carried out on RNA purifed from

RNA wass successfully purified from both the 1-week-old and 1-year-old FFPE tissues, includingly degraded small RNA fragments (Figure 1). Major RNA in the FFPE tissues agented after 1 year of storage, as astred by the reduced size of the size of the RNA and the oses of the rRNA double peaks in the agilent bioanalyzer traces. RNA purified from all tissues yielded A260/A280 ratios s of around 2.0, an director of high purity.

RNA development: RNA was purified from the tissue samples after 1 week or 1 year of storage. For the FFPE tissues, RNA was purified from 1-4 sections of of 10 sm thickness using the RNeasy FFPE Kit.

sections were cut on a standard microtome and the first 2 sections were discarded to exclude the effects of air exposure. For the RNAlater stabilized tissues, RNA was purified using the RNeasy Lipid Tissue Mini Kit. RNA was quantited by measuring absorbance at 260 nm on a nanoDrop® spectrophotometer. RNA quality was assessed on an Agilent® bioanalyzer in "Eukaryotic Total RNA Nano" mode.