GE Healthcare

illustra
RNAspin Mini RNA Isolation
Kit
Product booklet
Codes: 25-0500-70 (20 preps)
25-0500-71 (50 preps)
25-0500-72 (250 preps)
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2
Page finder
1. Legal 3
2. Handling 4
2.1. Safety warnings and precations 4
2.2. Storage conditions 4
2.3. Expiry 4
3. Components 5
3.1. Kit contents 5
3.2. Reagents to be supplied by user 5
4. Description 6
4.1. The basic principle 6
4.2. Kit specifications 7
5. Preparation of working solutions 11
5.1. RNase-free DNase I 11
5.2. Buffer RA3 11
6. Handling, preparation, and storage of starting materials 12
7. Protocols 14
7.1. Total RNA purification from cultured cells and tissue
with RNAspin Mini Kit 14
7.2. Support protocol RNAspin Mini: Total RNA preparation
from non-blood biological fluids (e.g., serum, culture medium) 18
7.3. Support protocol RNAspin Mini: Total RNA preparation
from up to 10
9
bacterial cells 18
7.4. Support protocol RNAspin Mini: Total RNA preparation
from up to 10
8
yeast cells 19
7.5. Support protocol—RNAspin Mini and RNAspin Midi:
Clean-up of RNA from reaction mixtures 20
8. Appendix 21
8.1. Troubleshooting 21
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1. Legal
Product use restriction
The RNAspin Mini Kit components have been designed, developed,
and sold for research purposes only. They are suitable for in
vitro uses only. No claim or representation is intended for its use
to identify any specific organism or for clinical use (diagnostic,
prognostic, therapeutic, or blood banking).
It is the responsibility of the user to verify the use of the RNAspin
Mini Kit for a specific application range as the performance
characteristic of this kit has not been verified to a specific organism.
GE and GE monogram are trademarks of General Electric Company.
© 2006 General Electric Company – All rights reserved.
GE Healthcare reserves the right, subject to any regulatory approval,
to make changes in specifications and features shown herein, or
discontinue the product described at any time without notice or
obligation.
Contact your GE Healthcare representative for the most current
information and a copy of the terms and conditions
http://www.gehealthcare.com
GE Healthcare UK Limited.
Amersham Place, Little Chalfont,
Buckinghamshire, HP7 9NA UK
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2. Handling
or eyes wash immediately
with water. See material safety
data sheet(s) and/or safety
statement(s) for specific advice.
2.2. Storage
Store lyophilized RNase-free
DNase I at +4ºC on arrival
(stable up to 1 year).
All other kit components should
be stored at room temperature
(20–25°C) and they are stable
for up to one year. Storage at
lower temperatures may cause
precipitation of salts.
2.3. Expiry
For expiry date please refer to
outer packaging label.
2.1. Safety warnings
and precautions
Warning: For research use
only.
Not recommended or intended
for diagnosis of disease in
humans or animals. Do not
use internally or externally in
humans or animals.
Buffers RA1, RA2 and
MDB contain guanidine
thiocyanate. Wear gloves and
safety glasses.
All chemicals should be
considered as potentially
hazardous. We therefore
recommend that this product
is handled only by those
persons who have been trained
in laboratory techniques and
that it is used in accordance
with the principles of good
laboratory practice. Wear
suitable protective clothing
such as laboratory overalls,
safety glasses and gloves.
Care should be taken to avoid
contact with skin or eyes. In
the case of contact with skin
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3. Components
3.1. Kit contents
*Reagents highlighted with asterisk require prior preparation.
Table 3.1. RNAspin Mini kit contents
Pack Size 20 preps 50 preps 250 preps
Cat. No. 25-0500-70 25-0500-71 25-0500-72
Buffer RA1 10 ml 25 ml 125 ml
Buffer RA2 15 ml 15 ml 80 ml
Buffer RA3 (concentrate)* 5 ml 12.5 ml 3 x 25 ml
Buffer MDB (Membrane
Desalting Buffer) 10 ml 25 ml 125 ml
DNase reaction buffer 3 ml 7 ml 35 ml
DNase I, RNase-free
(lyophilized)* 1 vial 1 vial 5 vial
H
2
O
(RNase-free) 5 ml 15 ml 65 ml
RNAspin Mini Filter units
(violet) 20 50 250
RNAspin Mini columns (light
blue-plus collection tube) 20 50 250
RNAspin Mini collection tubes 60 150 750
1.5 ml microcentrigue tubes 20 50 250
Protocol 1 1 1
3.2. Reagents to be supplied by user
70% and 95–100% ethanol
β-mercaptoethanol
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4. Description
4.1 The basic principle
Fig 4.1. Overview of the RNAspin Mini procedure
Figure 4.1. shows an overview of an RNA isolation procedure using
the RNAspin Mini Kit. One of the most important aspects in the
isolation process is to prevent the degradation of the RNA during
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the isolation procedure. With the RNAspin Mini method, cells are
lysed by incubation in a solution containing large amounts of
chaotropic ions. This lysis buffer immediately inactivates RNases,
which are present in virtually all biological materials, and creates
appropriate binding conditions that favor adsorption of RNA to the
silica membrane. Contaminating DNA, which is also bound to the
silica membrane, is removed by the direct application of a DNase
I solution to the silica membrane (RNase-free DNase I is supplied
with the kit). Simple washing steps with two different buffers remove
salts, metabolites and macromolecular cellular components. Finally,
pure RNA is eluted under low ionic strength conditions with RNase-
free water (supplied).
RNA isolation using the RNAspin Mini Kit can be performed at
room temperature. However, the eluate should be treated with care
because RNA is very sensitive to trace contaminations of RNases,
often found on general labware, fingers and dust. To preserve
stability, keep the isolated total RNA frozen at -20°C for short-term
or -80°C for long-term storage.
4.2 Kit specifications
The RNAspin Mini Kit is recommended for the isolation of total RNA
from cultured cells and tissue. The kit can be used to isolate RNA
from different amounts of sample material according to Table 4.1.
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Table 4.1. Examples of Input Sample Material
Sample amount
Cultured animal cells
(e.g., HeLa cells) up to 5 x 10
6
Animal tissue up to 30 mg
Bacteria up to 1 x 10
9
Yeast up to 5 x 10
7

RNAspin Mini Kits allow for the isolation of pure RNA with an
A
260
/A
280
ratio generally exceeding 1.9 (measured in 0.1 M Tris-HCl
buffer (pH 7.6)). Even biological samples that are sometimes difficult
to process, will yield high quality RNA. These include mouse tissue
(liver, brain), different tumor cell lines, Streptococci, and Actinobacillus
pleuropneumoniae. Note that this kit is not suitable for isolation of
RNA from blood.
The isolated RNA is ready to use for downstream applications like
quantitative Reverse Transcriptase-PCR (QRT-PCR), Primer Extension,
RNase Protection Assays, cDNA Synthesis and Microarray Analysis.
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A B
28S
18S
Fig. 4.2 The RNAspin Mini kits produce high quality RNA: rRNA
bands are sharp, with the 28S band being about twice as intense as
the 18S band, and with good RNA Integrity Number (RIN) values. (A)
Total RNA from 10
6
HeLa cells was isolated with RNAspin Mini and
separated by gel electrophoresis on a 1.2% formaldehyde agarose
gel; (B) Total RNA from rat liver was isolated with RNAspin Mini and
evaluated using the Agilent 2100 bioanalyzer.
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Table 4.2. RNAspin Mini Technical Specifications at a Glance
Animal tissue Cell culture
Sample size up to 30 mg tissue up to 5 x 10
6
cells
Typical yield up to 70 µg up to 70 µg
Elution volume 40–120 µl
Effective binding
capacity 100 µg
RNA integrity sharp rRNA bands with no substantial
degradative bands visible
28S:18S = ~2:1
RNA Integrity Number (RIN) values ≥7
RNA purity A
260
/A
280
= 1.8–2.2
Time/Prep <30 min/6 preps
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5. Preparation of working solutions
5.1. RNase-free DNase I
Avoid vigorous mixing of the DNase I enzyme because it is sensitive
to mechanical agitation.
Add the indicated volume of RNase-free water to the DNase I vial
and incubate for 1 min at room temperature. Gently swirl the vials
to completely dissolve the DNase I. Dispense into aliquots and store
at -20°C. The frozen working solution is stable for 6 months. Do not
freeze/thaw the aliquots more than three times.
5.2. Buffer RA3
Add the indicated volume of 96–100% ethanol to the RA3
concentrate. Store buffer RA3 at room temperature (20–25°C) for up
to one year.
Table 5.1. Reagent Preparation
20 preps 50 preps 250 preps
Cat. No. 25-0500-70 25-0500-71 25-0500-72
Buffer RA3 5 ml 12.5 ml 3 x 25 ml
(concentrate) add 20 ml add 50 ml add 100 ml
ethanol ethanol ethanol to
each bottle
DNase I, RNase - 1 vial 1 vial 5 vials
free (lyophilized) add 230 µl add 540 µl add 540 µl
RNase-free RNase-free RNase-free
water water water
to each vial
11
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6. Handling, preparation, and storage
of starting materials
RNA is not protected against digestion until the sample material
is flash frozen or disrupted in the presence of RNase inhibiting or
denaturing agents. Therefore it is important that samples are flash
frozen in liquid N
2
immediately, and stored at -70°C, stored in a
stabilizing agent, or processed as soon as possible. Samples can
be stored in lysis buffer RA1 after disruption at -70°C for up to one
year, at +4°C for up to 24 hours or up to several hours at room
temperature. Frozen samples are stable up to 6 months. Frozen
samples in buffer RA1 should be thawed slowly before starting with
the isolation of total RNA.
Wear gloves at all times during the preparation. Change gloves
frequently.
Cultured animal cells are collected by centrifugation and directly
lysed by adding buffer RA1 according to step 2 of the standard
protocol.
Animal tissues are often solid and must therefore be broken up
mechanically as well as lysed. Depending on the disruption method,
the viscosity of the lysed sample has to be reduced further for
optimal results. It is essential for efficient RNA preparation that
all the RNA contained in the sample is released from the cells
by disruption and that the viscosity of the sample is reduced by
homogenization.
The most commonly used technique for disruption of animal tissues
is grinding with a pestle and mortar. Grind the sample to a fine
powder in the presence of liquid N
2
. Take care that the sample
does not thaw during or after grinding or weighing and add the
frozen powder to an appropriate aliquot of buffer RA1 containing
β-mercaptoethanol and mix immediately. The broken-up tissue must
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then be homogenized with a RNAspin Filter unit or by passing ≥5
times through a 0.9 mm syringe needle.
Thawing of undisrupted animal tissue should be exclusively done
in the presence of buffer RA1 and β-mercaptoethanol during
simultaneous mechanical disruption, e.g., with a rotor-stator
homogenizer. This ensures that the RNA is not degraded by RNases
before the preparation has started. The spinning rotor disrupts and
simultaneously homogenizes the sample by mechanical shearing of
DNA within seconds up to minutes (homogenization time depends
on sample). Take care to keep the rotor tip submerged in order to
avoid excess foaming. Select a suitably sized homogenizer (5–7 mm
diameter rotors can be used for homogenization in microcentrifuge
tubes).
Bacteria and yeasts have to be incubated in lysozyme or lyticase/
zymolase solutions, respectively (see support protocols in section
7). By this treatment, the robust cell walls of these organisms are
digested or at least weakened, which is essential for effective cell
lysis by buffer RA1. For microorganisms with extremely resistant
cell walls – like some Gram-positive bacterial strains – it may be
necessary to optimize the conditions of the treatment with lytic
enzymes or the cultivation conditions. After lysis, homogenization is
achieved by the use of a RNAspin Filter units or the syringe-needle
method.
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1. Homogenization of sample
Disrupt up to 30 mg of tissue (for homogenization
methods and sample amounts see section 6).
Up to 5 x 10
6
eukaryotic cultured cells are
collected by centrifugation and lysed by addition
of buffer RA1 directly.
If not already homogenized, to process greater
amounts of cells (>1 x 10
6
) or tissue (>10 mg), first
homogenize using a 0.9 mm needle (20 gauge),
followed by filtration through RNAspin Mini Filter
units.
2. Cell lysis
Add 350 µl buffer RA1 and 3.5 µl
β-mercaptoethanol to the cell pellet or to ground
tissue and vortex vigorously
3. Filtration of the lysate
Reduce viscosity and clear the lysate by filtration
through RNAspin Mini Filter units: Place RNAspin
Mini Filter units (violet ring) in a collection tube,
apply the mixture, and centrifuge for 1 min at
11 000 x g.
Alternatively, the lysate may be passed ≥ 5 times
through a 0.9 mm needle (20 gauge) fitted to a
syringe.
14
disrupt
sample
+ 350 µl RA1
+ 3.5 µl β-ME
1 min
11 000 x g
7. Protocols
7.1. Total RNA purification from cultured cells
and tissue with RNAspin Mini Kit
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Discard the RNAspin Mini Filter. Transfer filtrate,
taking care to avoid aspirating any formed
pellet, to a new 1.5 ml microcentrifuge tube (not
included).
4. Adjust RNA binding conditions
Add 350 µl ethanol (70%) to the homogenized
lysate and mix by vortexing, 2 x 5 sec.
After the addition of ethanol, a stringy precipitate
may become visible. This will not affect RNA
isolation. Be sure to load all of the precipitate onto
the column as described in step 5.
5. Bind RNA
For each preparation, use one RNAspin Mini
column (light blue ring) placed in a 2 ml
microcentrifuge tube. Pipet lysate up-and-down
2–3 times, and then load the lysate onto the
column. Centrifuge for 30 s at 8000 x g. Place the
column in a new collection tube.
Maximal loading capacity of RNAspin Mini column
is 750 µl. Repeat the procedure if larger volumes
are to be processed.
6. Desalt silica membrane
Add 350µl MDB (Membrane Desalting Buffer)
and centrifuge at 11 000 x g for 1 min to dry the
membrane. Discard the flow-through and return
the column to the collection tube.
Salt removal will make the subsequent DNase I
digest much more effective. If the column outlet
has come into contact with the flow-through
+ 350 µl
70% EtOH
mix
load lysate
30 sec
8000 x g
+350 µl MDB
1 min
11 000 x g
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for any reason, discard the flow-through and
centrifuge again for 30 sec at 11 000 x g.
7. Digest DNA
Prepare DNase reaction mixture in a sterile
microcentrifuge tube: for each isolation, add 10µl
reconstituted DNase I (from section 5) to 90µl
DNase reaction buffer. Mix by flicking the tube.
Apply 95 µl of DNase reaction mixture directly
onto the center of the silica membrane of the
column. Incubate at room temperature for 15
min.
8. Wash and Dry silica membrane
Make sure buffer RA3 is equilibrated to room
temperature.
1st wash
Add 200µl buffer RA2 to the RNAspin Mini column.
Centrifuge for 1 min at 11 000 x g. Place the
column into a new collection tube.
Buffer RA2 will inactivate DNase I.
2nd wash
Add 600 µl buffer RA3 to the RNAspin Mini
column. Centrifuge for 1 min at 11 000 x g.
Discard flow-through and place the column back
into the collection tube.
16
+ 95 µl DNase
reaction
mixture
RT
15 min
+ 200 µl RA2
1 min
11 000 x g
+600 µl RA3
1 min
11 000 x g
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3rd wash
Add 250 µl buffer RA3 to the RNAspin Mini
column. Centrifuge for 2 min at 11 000 x g to
dry the membrane completely. Place the column
into a nuclease-free 1.5 ml microcentrifuge tube
(supplied).
If for any reason the liquid level in the collection
tube has reached the RNAspin Mini column
after centrifugation, discard flow-through and
centrifuge again.
Check that no residual flow-through remains in
the column outlet. If any remains, re-centrifuge.
9. Elute highly pure RNA
Elute the RNA in 100 µl H
2
O (RNase-free; supplied)
and centrifuge at 11 000 x g for 1 min.
It is possible to adapt the elution protocol for the
subsequent application of interest.
High yield: Perform two elution steps with the
volume indicated in the individual protocol. About
90–100% of bound nucleic acid will be eluted.
High concentration: Elute with a 40 µl elution
volume.
High yield and high concentration: Elute with the
standard elution volume and apply the eluate a
second time onto the column for re-elution.
Eluted RNA should be immediately placed on ice
to prevent potential degradation. Keep at
-20°C and -80°C for short-and long-term storage,
respectively.
+250 µl RA3
2 min
11 000 x g
+ 100 µl H
2
O
(RNase-free)
1 min
11 000 x g
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7.2. Support protocol RNAspin Mini: Total RNA
preparation from non-blood biological fluids
(e.g., serum, culture medium)
No need for homogenization or filtration of the lysate.
1. Cell lysis
Add 350 µl buffer RA1 to 100 µl of sample and vortex vigorously .
2. Adjust RNA binding conditions
Add 350 µl of ethanol (70%) to the lysate and mix by vortexing, 2 x
5 sec
Proceed with step 5 of the RNAspin Mini standard protocol (section
7.1).
7.3. Support protocol RNAspin Mini: Total RNA
preparation from up to 10
9
bacterial cells
1. Homogenization of sample
Resuspend the bacterial cell pellet (Gram-negative strains) in 100 µl
TE buffer (10 mM Tris-HCl, 1 mM EDTA pH 8) containing 0.2 mg/ml
lysozyme by vigorous vortexing. Incubate at 37°C for 10 min.
For preparation of RNA from Gram-positive bacteria, resuspend cells
in 100 µl TE containing 2 mg/ml lysozyme. It may be necessary to
optimize incubation time and lysozyme concentration, according to
the bacterial strain.
2. Cell lysis
Add 350 µl buffer RA1 and 3.5 µl β-mercaptoethanol to the
suspension and vortex vigorously.
3. Filtration of lysate
Reduce viscosity and turbidity of the solution by filtration through
RNAspin Mini Filter units. Place the RNAspin Mini Filter units in
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collection tubes, apply mixture, and centrifuge for 1 min at 11 000
x g.
Alternatively, the lysate may be passed ≥ 5 times through a 0.9 mm
needle (20 gauge) fitted to a syringe.
4. Adjust RNA binding conditions
Add 350 µl of ethanol (70%) and proceed with step 5 of the RNAspin
Mini standard protocol (section 7.1).
Because of the higher relative concentration of genome equivalents
in a nucleic acid preparation of bacteria compared with eukaryotic
material, it may be necessary to use a lower amount of cells for the
preparation.
7.4. Support protocol RNAspin Mini: Total RNA
preparation from up to 5 x 10
8
yeast cells
1. Homogenization of sample
Harvest 2–5 ml of YPD culture (centrifugation at 5000 x g, 10 min).
Decant culture media. Resuspend pellet in sorbitol/lyticase buffer
(50–100 U lyticase or zymolase in 1 M sorbitol/100 mM EDTA) and
incubate at 30°C for 30 min. Pellet the resulting spheroplasts by
centrifugation (1000 x g, 10 min).
It may be necessary to optimize incubation time and lyticase/
zymolase concentration, according to the yeast strain.
2. Cell lysis
Add 350 µl buffer RA1 and 3.5 µl β-mercaptoethanol to the
suspension and vortex vigorously.
3. Filtration of lysate
Reduce viscosity and turbidity of the solution by filtration through
RNAspin Mini Filter units. Place RNAspin Mini Filter units in collection
tubes, apply mixture, and centrifuge for 1 min at 11 000 x g.
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20
Alternatively, the lysate may be passed ≥5 times through a 0.9 mm
needle (20 gauge) fitted to a syringe.
Proceed with step 4 of the RNAspin Mini standard protocol (section
7.1)
Due to the higher relative concentration of genome equivalents in a
nucleic acid preparation of yeasts compared with cultured cells or
tissue material, it may be necessary to use a lower amount of cells
for the preparation.
7.5. Support protocol - RNAspin Mini and
RNAspin Midi: Clean-up of RNA from reaction
mixtures
1. Clean-up
Add 3.5 volumes of buffer RA1 per 1 volume of sample, and vortex
to mix.
Maximal loading capacity of RNAspin Mini columns is 750 µl. Repeat
the procedure if larger volumes are to be processed.
In order to load all of the sample in subsequent step 5 in one step:
- do not use more than 90 µl of sample for the RNAspin Mini Kit,
- do not use more than 480 µl of sample for the RNAspin Midi Kit.
2. Adjust RNA binding conditions
Add ethanol (95–100%) at 3.5 times the volume of sample only
from step 1 to the lysate, and mix by vortexing.
Proceed with step 5 of section 7.1 of either the RNAspin Mini or the
RNAspin Midi standard protocols.
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21
8. Appendix
8.1. Troubleshooting guide
Problem: RNA is degraded/no RNA obtained
Possible cause Suggestions
Problem: Poor RNA quality or yield
Possible cause Suggestions
RNase contamination
Reagents not
prepared, stored or
applied properly
• Create an RNase-free working
environment. Wear gloves during
all steps of the procedure, and
change gloves frequently. Use of
sterile, disposable polypropylene
tubes is recommended. Keep tubes
closed whenever possible during the
preparation. Glassware should be oven-
baked for at least 2 hours at 250°C
before use.
Reagents not properly stored. Add the
indicated volume of nuclease-free water
to DNase I vial and 96% ethanol to buffer
concentrate RA3 and mix. Reconstitute
and store lyophilized DNase I according
to instructions given in section 5.
• Sample and reagents have not been
mixed completely. Always vortex
vigorously after each reagent has been
added.
• Ethanol was not added after lysis.
Binding of RNA to the silica membrane is
only effective in the presence of ethanol.
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Problem: Poor RNA quality or yield
Possible cause Suggestions
Reagents not
prepared, stored or
applied properly,
continued
Suboptimal elution
Sample material
• Reconstitute and store lyophilized
DNase I according to instructions given
in section 5.
• Store other kit components at room
temperature. Storage at low tempera-
tures may cause salt precipitation.
• Keep bottles tightly closed in order to
prevent evaporation or contamination.
• Be sure that all of the water gets into
contact with the silica membrane. No
water drops should stick to the walls of
the columns. The membrane has to be
wetted completely.
Ionic strength and pH influence A
260

absorption as well as ratio A
260
/A
280
;
thus, for absorption measurement, use
0.1 M Tris-HCl pH 7.6 as diluent.
• Sample material not stored properly.
Whenever possible, use fresh material.
If this is not possible, flash freeze the
samples in liquid N
2
or treat with a
stabilizing agent. Samples should always
be kept at -80°C. Never allow tissues
to thaw before addition of buffer RA1.
Perform disruption of samples in liquid
N
2
, if possible.
22
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23
Problem: Poor RNA quality or yield. continued
Possible cause Suggestions

Problem: Clogged RNAspin Binding Column
Possible cause Suggestions
Sample material,
continued
Sample material
• Insufficient disruption and/or
homogenization of starting material.
Ensure thorough sample disruption and
use RNAspin Mini Filter for easy clean-up
of disrupted starting material.
• Too much starting material may
lead to RNA Binding column clogging
or reduced RNA quality or yield. For
clogging issues, see below. RNA quality
and yield problems relating to too much
sample material may be addressed
by decreasing the amount of starting
material and/or increasing the volumes
of wash buffers RA2 and RA3.
• Use the RNAspin Mini Filter to reduce
the risk of clogging the Binding column.
• To prevent clogging due to too much
starting material, reduce the sample
amount, increase the time for the
centrifugation steps, and/or increase
the volume of buffer RA1. If clogging
still occurs during the run, take the
remaining lysate off the RNAspin RNA
Binding column, discard it, and proceed
with the desalting step (with buffer MDB).
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Problem: Contamination of RNA with genomic DNA
Possible cause Suggestions
DNase I not active
DNase solution not
properly applied
Too much cell material
used
DNA detection system
too sensitive
• Reconstitute and store lyophilized
DNase I according to instructions given
in section 5.
• Pipette DNase I solution directly onto
the center of the silica membrane.
• Reduce quantity of cells or tissue used
• The amount of DNA contamination
is significantly reduced during the on-
column DNase I digestion. Residual
DNA may still be present; therefore in
very sensitive applications, it might be
possible to detect DNA.
As part of the product QC process, the
RNAspin Mini system is assessed for
DNA contamination by the following
procedure:
1 x 10
6
HeLa cells are subjected to RNA
isolation according to the protocol.
RNA eluate is used as template for PCR
detection of a 1 kb fragment in a 30-
cycle reaction.
Generally, no PCR fragment is obtained
if DNase I is applied, however, a strong
PCR fragment is obtained if the DNase is
omitted.
24
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25
Problem: Contamination of RNA with genomic DNA, continued
Possible cause Suggestions
Problem: Suboptimal performance of RNA in downstream
experiments
Possible cause Suggestions
DNA detection system
too sensitive
continued
Carry-over of ethanol
or salt
Store isolated RNA
properly
The potential for DNA contamination
detection by PCR increases with:
• the number of DNA copies per
preparation:
single copy target < plastidial/mitochondrial target
< plasmid transfected into cells
• decreasing PCR amplicon size: use
larger PCR targets (e.g. >500 bp) or
intron-spanning primers if possible.
• Do not let the flow-through touch
the column outlet after the second
RA3 wash. Be sure to centrifuge at the
corresponding speed for the respective
time in order to remove ethanolic buffer
RA3 completely.
• Check if buffer RA3 has been
equilibrated to room temperature before
use. Washing at lower temperatures
lowers efficiency of salt removal by RA3.
• Eluted RNA should always be kept
on ice for optimal stability since trace
contaminations of omnipresent RNases
will degrade the isolated RNA. For short-
term storage freeze at -20°C, for long-
term storage, freeze at -80°C.
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26
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http://www.gehealthcare.com/lifesciences
GE Healthcare UK Limited
Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA
UK
GE Healthcare
regional office
contact numbers:
Asia Pacific
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25-0500-70PL Rev A 2006
25-0500-70PL final.indd 28 18/5/06 08:26:20
The next two pages are a
protocol card.
Please add to the back page as a
tear off addition.
25-0500-70PL final.indd 29 18/5/06 08:26:21
illustra
RNAspin Mini RNA Isolation Kit
Product protocol card 25-0500-70
1. Homogenization of sample
2. Cell lysis
3. Filtration of the lysate
4. Adjust RNA binding conditions
5. Bind RNA
6. Desalt silica membrane
7. Digest DNA
<30 mg
+ 350 µl RA1
+ 3.5 µl β-ME
mix
1 min
11 000 x g
+ 350 µl
70% EtOH
mix
load lysate
30 sec
8,000 x g
+ 350 µl MDB
1 min
11 000 x g
+ 95 µl DNase
reaction mixture
RT
15 min
25-0500-70PL final.indd 30 18/5/06 08:26:21
Product use restriction
The RNAspin Mini Kit components have been designed, developed, and sold for research
purposes only. They are suitable for in vitro uses only. No claim or representation
is intended for its use to identify any specific organism or for clinical use (diagnostic,
prognostic, therapeutic, or blood banking).
It is the responsibility of the user to verify the use of the RNAspin Mini Kit for a specific
application range as the performance characteristic of this kit has not been verified to a
specific organism.GE and GE monogram are trademarks of General Electric Company.
GE and GE monogram are trademarks of General Electric Company.
© 2006 General Electric Company – All rights reserved.
GE Healthcare reserves the right, subject to any regulatory approval, to make changes in
specifications and features shown herein, or discontinue the product described at any time
without notice or obligation.
Contact your GE Healthcare representative for the most current information and a copy of
the terms and conditions
http://www.gehealthcare.com/lifesciences
GE Healthcare UK Limited
Amersham Place Little Chalfont
Buckinghamshire HP7 9NA UK
8. Wash and dry silica membrane
9. Elute highly pure RNA

1
st
wash.
+ 200 µl RA2
2
nd
wash
+ 600 µl RA3
3
rd
wash
+ 250 µl RA3
1
st
and 2
nd
wash
1 min
11 000 x g
3
rd
wash
2 min
11 000 x g
+ 100 µl H
2
O
(RNase-free)
1 min
11 000 x g
25-0500-70PC Rev A 2006
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