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Obtaining RNA from fibrous tissue is difficult due to contractile proteins, connective tissue, and collagen, which can all
interfere with the isolation process. To remove these proteins, the sample needs to be treated with a protease or phenol
containing lysis reagents. However, this requires conditions that do not degrade RNA, such as with an RNasefree
proteinase K digest.
Isolating RNA from heart, muscle, and other fibrous tissue
Friederike Krämer, Abhishek SharmaQIAGEN GmbH, QIAGEN Strasse 1, 40724 Hilden, Germany
Obtaining RNA from FFPE samples
Isolating RNA from human blood samples Stabilizing RNA in collected blood samples
Removing genomic DNA
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Special considerations for RNA isolation from different sample sources – Part I
Sample to Insight
RNA isolated from various fibrous tissues. RNA was isolated using the RNeasy® Fibrous Tissue Mini Kit. Realtime, quantitative RTPCR was performed with 100 ng total RNA using the QuantiTect® Probe RTPCR Kit and primers and probe specific for alphaactin.
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DNA
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Liver
RNeasy PlusSupplier AIV
Kidney Lung
Supplier I
Genomic DNA contamination of the isolated RNA significantly affects the interpretation of results of downstream
applications, such as RTPCR. RNasefree DNase or RNeasy gDNA Eliminator Columns are effective solutions.
Effective genomic DNA removal. Total RNA was purified in duplicate from various mouse tissues using the RNeasy Plus Mini Kit and kits from other suppliers. Realtime PCR assays for cjun were performed to determine the amount of gDNA contamination in the purified RNA.
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AllPrep FFPEQIAamp FFPE
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Both RNA and DNA can be obtained from formalinfixed, paraffinembedded (FFPE) tissue. Specific protocols exist to allow
molecular analysis of this valuable and extensive source of research material.
The fixation and embedding procedures heavily fragment and chemically modify nucleic acids, so they are often of a lower
molecular weight than those obtained from fresh or frozen samples. The degree of fragmentation and modification depends
on the type and age of the sample, and on the fixation, embedding, and storage conditions.
Minimize the effects of FFPE storage on RNA transcripts with the following tips:
• Remove and fix tissue as quickly as possible
• Use tissue samples no more than 5 mm thick and do not overfix (max. 24 hours)
• Use highquality reagents for paraffin embedding, without additives
• Avoid sample staining, if possible
• Store FFPE samples appropriately
Note: RNA remains intact for up to 1 year when stored at 4ºC
• Use an appropriate deparaffinization step
• Have a crosslinkreversal step during RNA isolation
Human erythrocytes do not contain nuclei or otherwise synthesize RNA and contain only very small amounts of RNA,
so the target of isolation from whole blood is leukocytes, which are nucleated. Removing the unwanted erythrocytes
simplifies RNA isolation. Selective lysis is an efficient method, because erythrocytes are more susceptible than leukocytes
to hypotonic shock.
A common alternative to erythrocyte lysis is Ficoll densitygradient centrifugation, which only recovers mononuclear
cells (lymphocytes and monocytes) and removes granulocytes. Mononuclear cells isolated in this way can then be
processed normally.
It is also essential to remove contaminants, such as the anticoagulants heparin and EDTA, and naturally occurring
enzyme inhibitors, which can all interfere with downstream RNA analysis. The QIAamp RNA Blood Mini Kit is highly
suited for RNA isolation from human blood as it removes RNases, contaminants, and enzyme inhibitors, with minimum
copurification of DNA.
Cellfree RNA in plasma, serum, and other body fluidsRNA, especially miRNA, associated with proteolipids (vesicles) or proteins can be found in the plasma, serum, urine,
and other body fluids, and in cell culture supernatants. The concentration is much lower than that of cellular RNA. It is
relatively stable, with a halflife of about 2 days in human whole blood. Nonetheless, it can be degraded by repeated
freeze–thaw cycles. Addition of carrier RNA may be necessary during isolation.
B CA
Isolation of DNA and RNA from FFPE tissues. A. DNA and B. RNA were purified from various FFPE rat tissues using the AllPrep® DNA/RNA FFPE Kit or the QIAamp® DNA FFPE Tissue Kit and the RNeasy FFPE Kit. The AllPrep Kit and the dedicated kits provided comparable CT values, indicating that all kits achieved similar efficiency in recovering usable DNA or RNA.C. Analysis of Jun expression in an FFPE sample of rat spleen tissue. The spleen is DNArich tissue. The sample was treated with the RNA isolation protocol of the AllPrep DNA/RNA FFPE Kit and the isolate was amplified with (+RT) and without (RT) reverse transcriptase. There results show an absence of DNA contamination.
Effective transcript stabilization. Blood samples (500 μl) were collected from a single rat into RNAprotect® Animal Blood Tubes or tubes containing EDTA. The samples were stored at 15–25ºC for the indicated times. RNA was purified using the RNeasy Protect Animal Blood Kit, and Fos expression was analyzed in duplicate using realtime RTPCR. ΔCT on the Yaxis shows CT values subtracted from those for the time = 0 samples. Fos expression remained stable in the RNAprotect stabilized samples, but changed with time in the EDTAtreated samples.
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For uptodate licensing information and productspecific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.qiagen.com or can be requested from QIAGEN Technical Services or your local distributor. Trademarks: QIAGEN®, QIAamp®, AllPrep®, QuantiTect®, RNAprotect®, RNeasy® (QIAGEN Group)
© QIAGEN 2015, all rights reserved.
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