Some sample sources present special challenges in RNA isolation or contain substances that cause problems in RNA analysis. These guides to RNA isolation have tips for a whole range of sample types, including guidance on the best kits for each.

1. General remarks on RNA and RNases
• Ribonucleases (RNases) are very stable and active enzymes that generally do not require cofactors to function,
and thus can easily degrade RNA in the lab.
• Do not use any plasticware or glassware without first eliminating possible RNase contamination.
• Great care should be taken to avoid inadvertently introducing RNases into the RNA sample during or after the
purification procedure.
• Purified RNA can be stored at –20ºC or –70ºC in RNase-free water. Under these conditions, no degradation of
RNA is detectable after 1 year.
General remarks on RNA and DNases
DNase digestion is not generally required for RNA isolated with RNeasy Kits since the silica gel membrane and spin column
technology efficiently removes the majority of the DNA. However, for certain RNA-based applications that are sensitive to
tiny amounts of DNA, combining the QIAGEN RNase-free DNase Set with the RNeasy protocol ensures pure RNA with no
DNA contamination.
Isolating RNA from bacteria
Bacterial mRNA has no 5’ cap and only rarely has a poly-A
tail. The absence of a poly-A tail means that mRNA isolation
by hybrid capture is impossible. In addition, oligo-dT
primers cannot be used to prime firststrand cDNA synthesis,
so random primers are needed.
Bacterial mRNA is also highly unstable, with an average
half-life of about 3 min for fast-growing bacteria. The mRNA
can degrade while it is still being translated. To accurately
preserve gene expression patterns and to maximize isolation
of fully intact mRNA, samples must be stabilized prior to
sample harvesting and processing.
The RNeasy Protect Bacteria Kit is makes bacterial gene
expression studies possible. It provides immediate in vivo
RNA stabilization and protection without using liquid
nitrogen, dry ice, or phenol. This allows the generation of
accurate gene expression profiles.
Some viruses have a single- or double-stranded RNA
genome. Viral RNA is typically isolated from cell-free body
fluids, where their titer can be very low. Virus particles
may need to be concentrated by ultracentrifugation,
ultrafiltration, or precipitation. Addition of carrier RNA may
also be necessary during RNA isolation when the expected
yield of RNA is low.
A major problem with viral RNA is that it typically has a high
degree of secondary structure. This can make downstream
analysis especially difficult. Many reverse transcriptases have
difficulty transcribing due to the complex RNA secondary
structure. In addition, RNA viruses have a high mutation
rate due to inaccurate copying when they replicate. This
can make it difficult to obtain a homogeneous population
for analysis.
0
20
40
60
80
– + – +
Supplier EIII
Genesexpressed(%)
RNeasy Protect
Bacteria
Rifampicin
102
103
104
105
106
– 1026 nt
CM M copies
Accurate gene expression profiles based on RNA from bacteria. Total RNA
was isolated from RNAprotect®
-stabilized E. coli cultures using the RNeasy
Protect Bacteria Kit or from unstabilized cultures using a commercial
precipitation method (Supplier EIII). To distinguish between gene expression
under defined culture conditions and the effects of artifactual gene induction
during harvesting and RNA isolation, the RNA polymerase inhibitor
rifampicin was added to half of the culture prior to RNA isolation.
Differences in transcript levels with and without rifampicin generally reflect
the degree of RNA degradation.
T A C M R
Isolating RNA from plant material
Friederike Krämer, Abhishek Sharma
QIAGEN GmbH, QIAGEN Strasse 1, 40724 Hilden, Germany
Isolating viral RNA
Discover RNeasy and QIAamp kits for RNA isolation
Reproducible purification of intact RNA from plant tissues
Special considerations for RNA isolation from different
sample sources – Part II
Sample to Insight
For up-to-date licensing information and product-specific 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®
, RNAprotect®
, RNeasy®
(QIAGEN Group)
© QIAGEN 2015, all rights reserved.
Find your RNA isolation solution at www.qiagen.com/RNA
Commonly used RNA isolation techniques often require adaptation for use with plant samples. Several plant metabolites,
such as polysaccharides, polyphenols, and flavones, have chemical properties that are similar to those of nucleic acids,
making them difficult to remove. Such copurified metabolites and contaminants introduced by the purification procedure
(such as salts or phenol) can inhibit enzymatic reactions or cause variations in UV spectrophotometric measurements and
gel migration.
RNA isolation is easier if the plants are grown under conditions that do not induce high levels of plant metabolites, but it is
difficult to make general statements about growth conditions because plants are so varied. As a general guideline, healthy,
young tissues are recommended. RNA yields from young tissues are often higher because young tissue generally contains
more cells and fewer metabolites than the same amount of older tissue.
Many “home-made” protocols RNA isolation recommend growing plants in darkness for 1 to 2 days before harvesting to
prevent high levels of plant metabolite accumulation.
Using a dedicated kit for isolating RNA from plant tissue, such as the RNeasy®
Plant Mini Kit, ensures that the plant
metabolites, contaminants, and genomic DNA are not copurified with the RNA.
Amplification of RNA from plasma. RT-PCR products of a 1026 nt RNA
fragment purified from plasma. Serial tenfold dilutions (as indicated) were
added to plasma and purified using the QIAamp®
Viral RNA Mini Kit.
M: markers; C: negative control.
RNA isolated from the leaves of five plant species. Frozen plant leaves were disrupted using the TissueLyser II (2 x 1 minute). RNA was purified using the
RNeasy Plant Mini Kit and analyzed on a 1.2 % formaldehyde agarose gel. The ribosomal RNA bands were sharp and of equal intensity, indicating
reproducible purification of intact RNA. T: tomato (100 mg); A: Arabidopsis (25 mg); C: cotton (100 mg); M: maize (100 mg); R: canola (100 mg).
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