Sample to Insight__
January 2018
RNeasy
®
Fibrous Tissue
Mini Handbook
For purification of total RNA
from heart, skeletal muscle,
aorta and other fiber-rich
tissues
RNeasy Fibrous Tissue Mini Handbook 01/2018
2
Contents
Handbook Revision History ......................................................................................... 3
Kit Contents .............................................................................................................. 4
Storage.................................................................................................................... 5
Intended Use ............................................................................................................ 5
Safety Information ..................................................................................................... 6
Quality Control ......................................................................................................... 6
Introduction .............................................................................................................. 7
Principle and procedure ................................................................................... 7
Equipment and Reagents to Be Supplied by User ........................................................... 9
Important Notes ...................................................................................................... 10
Determining the amount of starting material ...................................................... 10
Handling and storing starting material ............................................................. 12
Disrupting and homogenizing starting material ................................................. 13
Protocol: Purification of Total RNA Using the RNeasy Fibrous Tissue Mini Kit ................... 15
Troubleshooting Guide ............................................................................................. 22
Appendix A: General Remarks on Handling RNA ........................................................ 25
Appendix B: Storage, Quantification, and Determination of Quality of RNA ................... 28
Appendix C: DNase Digestion of RNA Eluates Using the RNase-Free DNase Set ............. 33
Ordering Information ............................................................................................... 35
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RNeasy Fibrous Tissue Mini Handbook 01/2018
Handbook Revision History
Document Changes Date
HB-0485-001 Initial release. October 2010
HB-0485-002 Changes to comply with GHS regulation, throughout the document. December 2014
HB-0485-003 Removal of RNeasy Fibrous Midi Kit (cat. no. 75742), including
associated protocols, general kit information (Table 1 and Table 2) and
ordering information.
Update into Sample-to-Insight branding and edit into revised style.
January 2018
RNeasy Fibrous Tissue Mini Handbook 01/2018
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Kit Contents
RNeasy Fibrous Tissue Mini Kit
Catalog no.
No. of preps
(50)
74704
50
RNeasy Mini Spin Columns (each in a 2 ml collection tube) 50
Collection Tubes (1.5 ml) 50
Collection Tubes (2 ml) 50
Proteinase K 2 ml
Buffer RLT* 45 ml
Buffer RW1* 45 ml
Buffer RPE† (concentrate) 11 ml
RNase-Free Water 4 x 10 ml
RNase-Free DNase Set:
RNase-Free DNase I (lyophilized)
Buffer RDD
RNase-Free Water
1500 units
2 x 2 ml
1.5 ml
Quick-Start Protocol 1
* Contains a guanidine salt. Not compatible with disinfectants containing bleach. See page 6 for safety information.
†
Before using for the first time, add 4 volumes of ethanol (96–100%) as indicated on the bottle to obtain a working
solution.
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RNeasy Fibrous Tissue Mini Handbook 01/2018
Storage
The RNeasy Fibrous Tissue Mini Kit is shipped at ambient temperature and should be stored
dry at room temperature (15–25°C). The RNase-Free DNase Set should be stored
immediately upon receipt at 2–8°C. All components are stable for at least 9 months under
these conditions.
The ready-to-use proteinase K included in the kit is dissolved in a specially formulated
storage buffer. The proteinase K is stable for up to 1 year after delivery when stored at room
temperature. To prolong the lifetime of the proteinase K, we recommend storage at 2–8°C.
Intended Use
The RNeasy Fibrous Tissue Mini Kit is intended for molecular biology applications. This
product is not intended for the diagnosis, prevention or treatment of a disease.
All due care and attention should be exercised in the handling of the products. We
recommend all users of QIAGEN products to adhere to the NIH guidelines that have been
developed for recombinant DNA experiments, or to other applicable guidelines.
RNeasy Fibrous Tissue Mini Handbook 01/2018
6
Safety Information
When working with chemicals, always wear a suitable lab coat, disposable gloves and
protective goggles. For more information, please consult the appropriate safety data sheets
(SDSs). These are available online in convenient and compact PDF format at
www.qiagen.com/safety where you can find, view and print the SDS for each QIAGEN kit
and kit component.
CAUTION
DO NOT add bleach or acidic solutions directly to the sample preparation
waste
Buffer RLT contains guanidine thiocyanate and Buffer RW1 contains a small amount of
guanidine thiocyanate. Guanidine salts can form highly reactive compounds when combined
with bleach. If liquid containing these buffers is spilt, clean with suitable laboratory detergent
and water. If the spilt liquid contains potentially infectious agents, clean the affected area
first with laboratory detergent and water, and then with 1% (v/v) sodium hypochlorite.
Quality Control
In accordance with QIAGEN’s ISO-certified Quality Management System, each lot of
RNeasy Fibrous Tissue Mini Kit is tested against predetermined specifications to ensure
consistent product quality.
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RNeasy Fibrous Tissue Mini Handbook 01/2018
Introduction
The RNeasy Fibrous Tissue Mini Kit is designed for optimal lysis of fiber-rich tissues and
purification of high-quality total RNA >200 nt.
Principle and procedure
Total RNA purification from fibrous tissues, such as skeletal muscle, heart and aorta tissue can
be difficult due to the abundance of contractile proteins, connective tissue and collagen. The
RNeasy Fibrous Tissue Mini Kit is supplied with proteinase K, which removes these proteins.
Tissue samples are first lysed in Buffer RLT and then diluted before being treated with
proteinase K. Debris is pelleted by centrifugation, and the supernatant is removed. The
supernatant is mixed with ethanol and then centrifuged through an RNeasy Mini spin column,
where RNA binds to the silica membrane. Traces of DNA that may co-purify with the RNA are
removed by DNase treatment on the silica membrane. DNase and any contaminants are
efficiently washed away, and high-quality total RNA is eluted in RNase-free water. The RNeasy
Fibrous Tissue Mini Kit purifies RNA from up to 30 mg tissue (Figure 1, next page).
With the RNeasy Fibrous Tissue Mini Kit, all RNA molecules longer than 200 nucleotides are
purified. The procedure provides enrichment for mRNA since most RNAs <200 nucleotides
(such as 5.8S rRNA, 5S rRNA, and tRNAs, which together comprise 15–20% of total RNA)
are selectively excluded. For purification of small RNA, including microRNA, from tissues
and cells, we recommend using miRNeasy Kits.
RNeasy Fibrous Tissue Mini Handbook 01/2018
8
Figure 1. RNeasy Fibrous Tissue Mini workflow.
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RNeasy Fibrous Tissue Mini Handbook 01/2018
Equipment and Reagents to Be Supplied by User
When working with chemicals, always wear a suitable lab coat, disposable gloves and
protective goggles. For more information, consult the appropriate safety data sheets (SDSs),
available from the product supplier.
14.3 M β-mercaptoethanol (β-ME) (commercially available solutions are usually 14.3 M)
or, as alternative, dithiothreitol (DTT)
Ethanol (96–100%)*
Sterile, RNase-free pipette tips
Disposable gloves
Water bath or heating block capable of reaching 55°C
Equipment for tissue disruption and homogenization (see page 12). We recommend
either the TissueRuptor II
®
with TissueRuptor Disposable Probes or the TissueLyser II (see
Ordering Information, page 35)
For stabilization of RNA in tissues (see “Handling and storing starting material”,
page 12): RNAlater
®
RNA Stabilization Reagent (cat. no. 76104 or 76106) or
Allprotect
®
Tissue Reagent (cat. no. 76405) or liquid nitrogen and dry ice
1.5 ml or 2 ml microcentrifuge tubes
Microcentrifuge (with rotor for 2 ml tubes)
* Do not use denatured alcohol, which contains other substances such as methanol or methylethylketone.
RNeasy Fibrous Tissue Mini Handbook 01/2018
10
Important Notes
Determining the amount of starting material
It is essential to use the correct amount of starting material in order to obtain optimal RNA
yield and purity. The maximum amount that can be used is determined by:
The type of tissue and its RNA content
The volume of Buffer RLT required for efficient lysis
The RNA binding capacity of the RNeasy Mini spin column
When processing samples containing high amounts of RNA, less than the maximum amount
of starting material shown in Table 1 (next page) should be used, so that the RNA binding
capacity of the RNeasy Mini spin column is not exceeded.
When processing samples containing low amounts of RNA, the maximum amount of starting
material shown in Table 1 can be used. However, even though the RNA binding capacity of
the RNeasy Mini spin column is not reached, the maximum amount of starting material must
not be exceeded. Otherwise, lysis will be incomplete and cellular debris may interfere with
the binding of RNA to the RNeasy Mini spin column membrane, resulting in lower RNA yield
and purity.
More information on using the correct amount of starting material is given in the protocols.
Table 2 (next page) shows expected RNA yields from various sources.
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RNeasy Fibrous Tissue Mini Handbook 01/2018
Table 1. RNeasy Mini spin column specifications
Specification RNeasy Mini spin column
Maximum binding capacity 100 µg RNA
Maximum loading volume 700 µl
Minimum elution volume 30 µl
Maximum amount of starting tissue ≤30 mg
Note: If the binding capacity of the RNeasy Mini spin column is exceeded, RNA yields will
not be consistent and may be reduced. If lysis of the starting material is incomplete, RNA
yields will be lower than expected, even if the binding capacity of the RNeasy Mini spin
column is not exceeded.
Table 2. Typical yields of total RNA
Mouse/rat tissue (10 mg) Yield of total RNA (µg)*
Heart 8–12
Aorta 8–12
Trachea 8–12
Esophagus 8–12
Muscle 5–10
Skin 4–8
Eye 8–12
Ear 10–15
* Amounts can vary due to factors such as species and developmental stage. Since the RNeasy Fibrous Tissue Mini Kit
enriches for mRNA and other RNA species >200 nucleotides, the total RNA yield does not include 5S rRNA, tRNA
and other low-molecular-weight RNAs, which make up 15–20% of total cellular RNA.
RNeasy Fibrous Tissue Mini Handbook 01/2018
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Handling and storing starting material
RNA in harvested tissue is not protected until the sample is treated with RNAlater RNA
Stabilization Reagent, flash-frozen or disrupted and homogenized in the presence of RNaseinhibiting or denaturing reagents. If unprotected, unwanted changes in the gene expression
profile will occur. It is therefore important that tissue samples are immediately frozen in liquid
nitrogen and stored at –90 to –65°C or immediately immersed in RNAlater RNA
Stabilization Reagent or AllProtect
®
Tissue Reagent at room temperature or immediately
frozen in liquid nitrogen and stored at –90 to –65°C. Otherwise, unwanted changes in the
gene expression profile will occur
The procedures for tissue harvesting and RNA protection should be carried out as quickly as
possible. Frozen tissue samples should not be allowed to thaw during handling or weighing.
After disruption and homogenization in Buffer RLT (lysis buffer), samples can be stored at –90
to –65°C for several months. Storage times differ for different tissue types and have not been
established for all possible tissues.
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RNeasy Fibrous Tissue Mini Handbook 01/2018
Disrupting and homogenizing starting material
Efficient disruption and homogenization of the starting material is an absolute requirement for
all total RNA purification procedures. Disruption and homogenization are two distinct steps:
Disruption: Complete disruption of plasma membranes of cells and organelles is
absolutely required to release all the RNA contained in the sample. Incomplete disruption
results in significantly reduced RNA yields.
Homogenization: Homogenization is necessary to reduce the viscosity of the lysates
produced by disruption. Homogenization shears high-molecular-weight genomic DNA
and other high-molecular-weight cellular components to create a homogeneous lysate.
Incomplete homogenization results in inefficient binding of RNA to the RNeasy Mini spin
column membrane and therefore significantly reduced RNA yields.
Disruption and homogenization of tissue samples can be carried out rapidly and efficiently
using either the TissueRuptor II (for processing samples individually) or the TissueLyser (for
processing multiple samples simultaneously). Disruption and homogenization with the
TissueRuptor II or TissueLyser generally results in higher RNA yields than with other methods.
Disruption and homogenization using the TissueRuptor II
The TissueRuptor II is a rotor–stator homogenizer that thoroughly disrupts and simultaneously
homogenizes single tissue samples in the presence of lysis buffer in 15–90 seconds,
depending on the toughness and size of the sample. The blade of the TissueRuptor
disposable probe rotates at a very high speed, causing the sample to be disrupted and
homogenized by a combination of turbulence and mechanical shearing.
For guidelines on using the TissueRuptor II, refer to the TissueRuptor II Handbook. For other
rotor–stator homogenizers, refer to suppliers’ guidelines.
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14
Disruption and homogenization using the TissueLyser II
In bead-milling, tissues can be disrupted by rapid agitation in the presence of beads and
lysis buffer. Disruption and simultaneous homogenization occur by the shearing and crushing
action of the beads as they collide with the cells. The TissueLyser II disrupts and homogenizes
up to 48 tissue samples simultaneously when used in combination with the TissueLyser
Adapter Set 2 x 24, which holds 48 x 2 ml microcentrifuge tubes containing stainless steel
beads of 5 mm mean diameter. For guidelines on using the TissueLyser II, refer to the
TissueLyser Handbook. For other bead mills, refer to suppliers’ guidelines.
Note: Tungsten carbide beads react with Buffer RLT and must not be used to disrupt and
homogenize tissues.
The TissueLyser II can also disrupt and homogenize up to 192 tissue samples simultaneously
when used in combination with the TissueLyser Adapter Set 2 x 96, which holds 192 x 1.2 ml
microcentrifuge tubes containing stainless steel beads of 5 mm mean diameter. In this case, we
recommend using the RNeasy 96 Universal Tissue Kit (cat. no. 74881), which provides highthroughput RNA purification from all types of tissue – including fiber-rich tissues – in 96-well
format.
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RNeasy Fibrous Tissue Mini Handbook 01/2018
Protocol: Purification of Total RNA Using the
RNeasy Fibrous Tissue Mini Kit
Determining the correct amount of starting material
It is essential to use the correct amount of tissue in order to obtain optimal RNA yield and
purity. A maximum of 30 mg tissue can generally be processed. For most tissues, the RNA
binding capacity of the RNeasy Mini spin column and the lysing capacity of Buffer RLT and
proteinase K will not be exceeded by this amount. Average RNA yields from various tissues
are given in Table 2 (page 11).
If there is no information about the nature of your starting material, we recommend starting
with no more than 10 mg tissue. Depending on RNA yield and purity, it may be possible to
use up to 30 mg tissue in subsequent preparations.
IMPORTANT: Do not overload the RNeasy Mini spin column, as this will significantly reduce
RNA yield and quality.
Weighing tissue is the most accurate way to quantify the amount of starting material. As a
guide, a 3 mm cube (27 mm
3
) of most animal tissues weighs 25–35 mg.
Important points before starting
If using the RNeasy Fibrous Tissue Mini Kit for the first time, read “Important Notes”
(page 10).
If working with RNA for the first time, read “Appendix A: General Remarks on Handling
RNA”, page 25).
If using the TissueRuptor II, ensure that you are familiar with operating it by referring to
the TissueRuptor II User Manual and TissueRuptor II Handbook.
RNeasy Fibrous Tissue Mini Handbook 01/2018
16
If using the TissueLyser II, ensure that you are familiar with operating it by referring to the
operating instructions and TissueLyser Handbook.
Since the RNase-inactivating Buffer RLT must be diluted to permit proteinase K digestion,
this protocol should not be used for tissues rich in RNases, such as pancreas or intestine.
Fresh, frozen, or RNAlater stabilized tissues can be used. If freezing tissues, flash-freeze in
liquid nitrogen and immediately transfer to –90 to –65°C, where they can be stored for
several months. Do not allow tissues to thaw during weighing or handling prior to
disruption in Buffer RLT. Homogenized tissue lysates from step 4 can also be stored at –90
to –65°C for several months. Incubate frozen lysates at 37°C in a water bath until
completely thawed and salts are dissolved before continuing with step 5. Avoid prolonged
incubation, which may compromise RNA integrity.
Important: Do not vortex reconstituted DNase I. DNase I is especially sensitive to
physical denaturation. Mixing should only be carried out by gently inverting the tube.
Buffer RLT may form a precipitate upon storage. If necessary, redissolve by warming, and
then place at room temperature (15–25°C).
Things to do before starting
β-Mercaptoethanol (β-ME) must be added to Buffer RLT before use. Add 10 µl β-ME per
1 ml Buffer RLT. Dispense in a fume hood and wear appropriate protective clothing.
Buffer RLT containing β-ME can be stored at room temperature for up to 1 month.
Alternatively, add 20 µl of 2 M dithiothreitol (DTT) per 1 ml Buffer RLT. The stock solution
of 2 M DTT in water should be prepared fresh, or frozen in single-use aliquots. Buffer RLT
containing DTT can be stored at room temperature for up to 1 month.
Buffer RPE is supplied as a concentrate. Before using for the first time, add 4 volumes of
ethanol (96–100%) as indicated on the bottle to obtain a working solution.
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RNeasy Fibrous Tissue Mini Handbook 01/2018
Prepare DNase I stock solution before using the RNase-Free DNase Set for the first time.
Dissolve the lyophilized DNase I (1500 Kunitz units) in 550 µl of the RNase-free water
provided in the RNase-Free DNase Set box. To avoid loss of DNase I, do not open the
vial. Inject RNase-free water into the vial using an RNase-free needle and syringe. Mix
gently by inverting the vial. Do not vortex.
For long-term storage of DNase I, remove the stock solution from the glass vial, divide it
into single-use aliquots, and store at –30 to –15°C for up to 9 months. Thawed aliquots
can be stored at 2–8°C for up to 6 weeks. Do not refreeze the aliquots after thawing.
Procedure
1. Heat a water bath or heating block to 55°C for proteinase K digestion in step 6.
2. If using the TissueLyser II, add one stainless steel bead (5 mm mean diameter) per 2 ml
microcentrifuge tube (not supplied). If working with tissues that are not stabilized in
RNAlater RNA Stabilization Reagent, place the tubes on dry ice.
3. Excise the tissue sample from the animal or remove it from storage. Determine the amount
of tissue. Do not use more than 30 mg. Proceed immediately to step 4.
Note: Weighing tissue is the most accurate way to determine the amount.
Note: If the tissue sample was stored in RNAlater RNA Stabilization Reagent, remove it
from the reagent using forceps and be sure to remove any excess reagent or crystals that
may have formed.
Note: RNA in harvested tissues is not protected until the tissues are treated with RNAlater
RNA Stabilization Reagent, flash-frozen or disrupted and homogenized in step 4. Frozen
tissues should not be allowed to thaw during handling. The relevant procedures should
be carried out as quickly as possible.
RNeasy Fibrous Tissue Mini Handbook 01/2018
18
4. Disrupt the tissue and homogenize the lysate using EITHER the TissueRuptor II (follow steps
5 and 6) OR the TissueLyser II (follow steps 7–12).
See “Disrupting and homogenizing starting material”, page 13, for more details on
disruption and homogenization.
Note: Ensure that β-ME is added to Buffer RLT before use (see “Things to do before
starting”, page 16).
Note: Incomplete homogenization leads to significantly reduced RNA yields and can
cause clogging of the RNeasy Mini spin column. Homogenization with the TissueRuptor II
or TissueLyser II generally results in higher RNA yields than with other methods.
Disruption and homogenization using the TissueRuptor II
5. Place the tissue in a suitably sized vessel. Add 300 µl Buffer RLT.
Generally, round-bottomed tubes allow more efficient disruption and homogenization
than conical-bottomed tubes.
6. Place the tip of the disposable probe into the vessel and operate the TissueRuptor II at full
speed until the lysate is uniformly homogeneous (usually 20–40 s). Proceed to step 13.
Note: To avoid damage to the TissueRuptor II and disposable probe during operation,
make sure the tip of the probe remains submerged in the buffer.
Disruption and homogenization using the TissueLyser II
7. Place the tissues in the tubes prepared in step 2.
8. If the tubes were stored on dry ice, place them at room temperature. Then immediately
add 300 µl Buffer RLT per tube.
9. Place the tubes in the TissueLyser Adapter Set 2 x 24.
10. Operate the TissueLyser II for 2 min at 20 Hz.
Note: The time depends on the tissue being processed and can be extended until the
tissue is completely homogenized.
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RNeasy Fibrous Tissue Mini Handbook 01/2018
11. Disassemble the adapter set, rotate the rack of tubes so that the tubes nearest to the
TissueLyser II are now outermost, and reassemble the adapter set. Operate the
TissueLyser II for another 2 min at 20 Hz.
Note: Rearranging the tubes allows even homogenization.
12. Carefully pipet the lysates into new microcentrifuge tubes (not supplied). Proceed to step 13.
Important: Do not reuse the stainless steel beads.
Preparation of total RNA
13. Add 590 µl RNase-free water to the lysate. Then add 10 µl proteinase K solution and mix
thoroughly by pipetting.
14. Incubate at 55°C for 10 min.
15. Centrifuge at 15–25°C for 3 min at 10,000 x g.
A small pellet of tissue debris will form, sometimes accompanied by a thin layer or film
on top of the supernatant.
16. Pipet the supernatant (approximately 900 µl) into a new 1.5 ml or 2 ml microcentrifuge
tube (not supplied).
Avoid transferring any of the pellet. If this is unavoidable, a small amount of pelleted
debris may be carried over without affecting the RNeasy procedure. Hold the pipette tip
under the thin layer or film on top of the supernatant, if present. This layer will usually
adhere to the outside of the pipette tip and should not be transferred.
17. Add 0.5 volumes (usually 450 µl) of ethanol (96–100%) to the cleared lysate. Mix well by
pipetting. Do not centrifuge.
Precipitates may be visible after addition of ethanol. This does not affect the procedure.
RNeasy Fibrous Tissue Mini Handbook 01/2018
20
18. Transfer 700 µl of the sample, including any precipitate that may have formed, to an
RNeasy Mini spin column placed in a 2 ml collection tube. Close the lid gently, and
centrifuge at 15–25°C for 15 s at ≥8000 x g (≥10,000 rpm). Discard the flow-through.*
Reuse the collection tube in step 19.
19. Repeat step 18 using the remainder of the sample. Discard the flow-through.*
Reuse the collection tube in step 20.
20. Add 350 µl Buffer RW1 to the RNeasy Mini spin column. Close the lid gently and
centrifuge at 15–25°C for 15 s at ≥8000 x g (≥10,000 rpm) to wash the membrane.
Discard the flow-through.
Reuse the collection tube in step 23.
Optional: If on-column DNase digestion is not desired, add 700 µl Buffer RW1 instead,
centrifuge for 15 s at ≥8000 x g, and discard the flow-through* (but not the collection
tube). Proceed to step 24.
21. Add 10 µl DNase I stock solution to 70 µl Buffer RDD. Mix by gently inverting the tube,
and centrifuge briefly to collect residual liquid from the sides of the tube.
Note: DNase I is especially sensitive to physical denaturation. Mixing should only be
carried out by gently inverting the tube. Do not vortex.
22. Add the DNase I incubation mix (80 µl) directly to the RNeasy Mini spin column
membrane and place on the benchtop (15–25°C) for 15 min.
Note: Be sure to add the DNase I incubation mix directly to the RNeasy Mini spin column
membrane. DNase digestion will be incomplete if part of the mix sticks to the walls or the
O-ring of the spin column.
23. Add 350 µl Buffer RW1 to the RNeasy Mini spin column. Close the lid gently and
centrifuge for 15 s at ≥8000 x g (≥10,000 rpm) at 15–25°C. Discard the flow-through.*
Reuse the collection tube in step 24.
* Flow-through contains Buffer RLT or Buffer RW1 and is therefore not compatible with bleach. See page 6 for safety
information.
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RNeasy Fibrous Tissue Mini Handbook 01/2018
24. Add 500 µl Buffer RPE to the RNeasy Mini spin column. Close the lid gently and
centrifuge at 15–25°C for 15 s at ≥8000 x g (≥10,000 rpm) to wash the membrane.
Discard the flow-through.
Reuse the collection tube in step 25.
Note: Buffer RPE is supplied as a concentrate. Ensure that ethanol is added to Buffer RPE
before use (see “Things to do before starting”, page 16).
25. Add 500 µl Buffer RPE to the RNeasy Mini spin column. Close the lid gently and
centrifuge at 15–25°C for 2 min at ≥8000 x g (≥10,000 rpm) to wash the membrane.
The long centrifugation dries the spin column membrane, ensuring that no ethanol is carried
over during RNA elution. Residual ethanol may interfere with downstream reactions.
Note: After centrifugation, carefully remove the RNeasy Mini spin column from the
collection tube so that the column does not contact the flow-through. Otherwise,
carryover of ethanol will occur.
26. Optional: Place the RNeasy Mini spin column in a new 2 ml collection tube (supplied)
and discard the old collection tube with the flow-through. Close the lid gently and
centrifuge at full speed for 1 min.
Perform this step to eliminate any possible carryover of Buffer RPE, or if residual
flow-through remains on the outside of the RNeasy Mini spin column after step 25.
27. Place the RNeasy Mini spin column in a new 1.5 ml collection tube (supplied). Add 30–50 µl
RNase-free water directly to the RNeasy Mini spin column membrane. Close the lid gently. To
elute the RNA, centrifuge for 1 min at ≥8000 x g (≥10,000 rpm) at 15–25°C.
28. Repeat step 27 using another 30–50 µl RNase-free water, or using the eluate from step
19 (if high RNA concentration is required). Reuse the collection tube from step 27. If the
expected RNA yield is >30 µg, there is no need to repeat step 28.
If using the eluate from step 27, the RNA yield will be 15–30% less than that obtained
using a second volume of RNase-free water, but the final RNA concentration will be higher.
RNeasy Fibrous Tissue Mini Handbook 01/2018
22
Troubleshooting Guide
This troubleshooting guide may be helpful in solving any problems that may arise. For more
information, see also the Frequently Asked Questions page at our Technical Support Center:
www.qiagen.com/FAQ/FAQList.aspx. The scientists in QIAGEN Technical Services are
always happy to answer any questions you may have about either the information and/or
protocols in this handbook or sample and assay technologies (for contact information, visit
www.qiagen.com).
Comments and suggestions
Clogged RNeasy Mini spin column
a) Inefficient disruption and/or
homogenization
See “Disrupting and homogenizing starting material” (page 13) for details on
disruption and homogenization methods.
Increase g-force and centrifugation time, if necessary.
In subsequent preparations, reduce the amount of starting material (see page
10 and protocol) and/or increase the homogenization time.
b) Too much starting material
In subsequent preparations, reduce the amount of starting material. It is essential
to use the correct amount of starting material (see page 10 and protocol).
c) Centrifugation temperature
too low
The centrifugation temperature should be 15–25°C. Some centrifuges may cool
to below 20°C even when set at 20°C. This can cause formation of precipitates
that can clog the RNeasy Mini spin column. If this happens, set the
centrifugation temperature to 25°C. Warm the ethanol-containing lysate to 37°C
before transferring it to the RNeasy Mini spin column.
Low RNA yield
a) Inefficient disruption and/or
homogenization
See “Disrupting and homogenizing starting material” (page 13) for details on
disruption and homogenization methods.
Increase g-force and centrifugation time, if necessary.
In subsequent preparations, reduce the amount of starting material (see page
10 and protocol) and/or increase the homogenization time.
b) Too much starting material
In subsequent preparations, reduce the amount of starting material. It is essential
to use the correct amount of starting material (see page 10 and protocol).
c) RNA still bound to RNeasy
Mini spin column membrane
Repeat RNA elution, but incubate the RNeasy Mini spin column on the benchtop
for 10 min with RNase-free water before centrifuging.
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RNeasy Fibrous Tissue Mini Handbook 01/2018
Comments and suggestions
d) Ethanol carryover
During the second wash with Buffer RPE (step 25), be sure to dry the RNeasy
Mini spin column membrane by centrifuging at ≥8000 x g (≥10,000 rpm) for
2 minutes at 15–25°C. After centrifugation, carefully remove the column from
the collection tube so that the column does not contact the flow-through.
Otherwise, carryover of ethanol will occur.
To eliminate any chance of possible ethanol carryover, place the RNeasy Mini
spin column in a new 2 ml collection tube and perform the optional 1-minute
centrifugation step as described in step 26 of the protocol.
Low or no recovery of RNA
RNase-free water incorrectly
dispensed
Add RNase-free water to the center of the RNeasy Mini spin column
membrane to ensure that the membrane is completely covered.
Low A
260/A280
value
Water used to dilute RNA for
A
260/A280
measurement
Use 10 mM Tris·Cl, pH 7.5, not RNase-free water, to dilute the sample before
measuring purity (see “Appendix B: Storage, Quantification, and
Determination of Quality of RNA”, page 28).
RNA degraded
a) Inappropriate handling of
starting material
For optimal results, ensure that tissue starting material samples are properly
stabilized and stored in RNAlater RNA Stabilization Reagent (for details, see
the RNAlater Handbook).
For frozen tissue samples, ensure that they were flash-frozen immediately in
liquid nitrogen and properly stored at –90 to –65°C. Perform the RNeasy
procedure quickly, especially the first few steps.
See “Appendix A: General Remarks on Handling RNA”, page 25 and “
Handling and storing starting material”, page 12.
b) RNase contamination
Although all RNeasy buffers have been tested and are guaranteed RNase-free,
RNases can be introduced during use. Be certain not to introduce any RNases
during the RNeasy procedure or later handling. See ““
Appendix A: General
Remarks on Handling RNA”, page 25.
Do not put RNA samples into a vacuum dryer that has been used in DNA
preparation where RNases may have been used.
DNA contamination in downstream experiments
No DNase treatment
Perform on-column DNase digestion using the RNase-Free DNase Set, as
described in the protocol. Alternatively, perform DNase digestion after RNA
purification. See “
Appendix C: DNase Digestion of RNA Eluates Using the
RNase-Free DNase Set”, page 33.
For real-time, two-step RT-PCR experiments, carry out the RT step using the
QuantiTect
®
Reverse Transcription Kit (cat. no. 205311), which provides cDNA
synthesis with integrated removal of genomic DNA contamination.
RNeasy Fibrous Tissue Mini Handbook 01/2018
24
Comments and suggestions
RNA does not perform well in downstream experiments
a) Salt carryover during elution
Ensure that Buffer RPE is at 15–25°C.
b) Ethanol carryover
During the second wash with Buffer RPE (step 25), be sure to dry the RNeasy
Mini spin column membrane by centrifuging at 8000 x g (10,000 rpm) for
2 min at 15–25°C. After centrifugation, carefully remove the column from the
collection tube so that the column does not contact the flow-through. Otherwise,
carryover of ethanol will occur.
To eliminate any chance of possible ethanol carryover, place the RNeasy Mini
spin column in a new 2 ml collection tube and perform the optional 1-minute
centrifugation step as described in step 26 of the protocol.
25
RNeasy Fibrous Tissue Mini Handbook 01/2018
Appendix A: General Remarks on Handling
RNA
Handling RNA
Ribonucleases (RNases) are very stable and active enzymes that generally do not require
cofactors to function. Since RNases are difficult to inactivate and even minute amounts are
sufficient to destroy RNA, 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. In order
to create and maintain an RNase-free environment, the following precautions must be taken
during pretreatment and use of disposable and nondisposable vessels and solutions while
working with RNA.
General handling
Proper microbiological, aseptic technique should always be used when working with RNA.
Hands and dust particles may carry bacteria and molds and are the most common sources
of RNase contamination. Always wear latex or vinyl gloves while handling reagents and
RNA samples to prevent RNase contamination from the surface of the skin or from dusty
laboratory equipment. Change gloves frequently and keep tubes closed whenever possible.
Keep purified RNA on ice when aliquots are pipetted for downstream applications.
Disposable plasticware
The use of sterile, disposable polypropylene tubes is recommended throughout the
procedure. These tubes are generally RNase-free and do not require pretreatment to
inactivate RNases.
RNeasy Fibrous Tissue Mini Handbook 01/2018
26
Nondisposable plasticware
Nondisposable plasticware should be treated before use to ensure that it is RNase-free.
Plasticware should be thoroughly rinsed with 0.1 M NaOH, 1 mM EDTA* followed by
RNase-free water (see ”Solutions”, page 27). Alternatively, chloroform-resistant plasticware
can be rinsed with chloroform* to inactivate RNases.
Glassware
Glassware should be treated before use to ensure that it is RNase-free. Glassware used for
RNA work should be cleaned with a detergent,* thoroughly rinsed, and oven baked at
240°C for at least 4 hours (overnight, if more convenient) before use. Autoclaving alone will
not fully inactivate many RNases. Alternatively, glassware can be treated with DEPC*
(diethyl pyrocarbonate). Fill glassware with 0.1% DEPC (0.1% in water), allow to stand
overnight (12 hours) at 37°C and then autoclave or heat to 100°C for 15 minutes to
eliminate residual DEPC.
Electrophoresis tanks
Electrophoresis tanks should be cleaned with detergent solution (e.g., 0.5% SDS),*
thoroughly rinsed with RNase-free water, and then rinsed with ethanol
†
and allowed to dry.
* When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For
more information, consult the appropriate safety data sheets (SDSs), available from the product supplier.
†
Plastics used for some electrophoresis tanks are not resistant to ethanol. Take proper care and check the supplier’s
instructions.
27
RNeasy Fibrous Tissue Mini Handbook 01/2018
Solutions
Solutions (water and other solutions) should be treated with 0.1% DEPC. DEPC is a strong,
but not absolute, inhibitor of RNases. It is commonly used at a concentration of 0.1% to
inactivate RNases on glass or plasticware or to create RNase-free solutions and water. DEPC
inactivates RNases by covalent modification. Add 0.1 ml DEPC to 100 ml of the solution to
be treated and shake vigorously to bring the DEPC into solution. Let the solution incubate for
12 hours at 37°C. Autoclave for 15 minutes to remove any trace of DEPC. DEPC will react
with primary amines and cannot be used directly to treat Tris* buffers. DEPC is highly
unstable in the presence of Tris buffers and decomposes rapidly into ethanol and CO
2
.
When preparing Tris buffers, treat water with DEPC first, and then dissolve Tris to make the
appropriate buffer. Trace amounts of DEPC will modify purine residues in RNA by
carbethoxylation. Carbethoxylated RNA is translated with very low efficiency in cell-free
systems. However, its ability to form DNA:RNA or RNA:RNA hybrids is not seriously affected
unless a large fraction of the purine residues have been modified. Residual DEPC must
always be eliminated from solutions or vessels by autoclaving or heating to 100°C for
15 minutes.
Note: RNeasy buffers are guaranteed RNase-free without using DEPC treatment and are
therefore free of any DEPC contamination.
* When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For
more information, consult the appropriate safety data sheets (SDSs), available from the product supplier.
RNeasy Fibrous Tissue Mini Handbook 01/2018
28
Appendix B: Storage, Quantification, and
Determination of Quality of RNA
Storage of RNA
Purified RNA may be stored at –30 to –15°C or –90 to –65°C in RNase-free water. Under
these conditions, no degradation of RNA is detectable after 1 year.
Quantification of RNA
The concentration of RNA should be determined by measuring the absorbance at 260 nm
(A
260
) in a spectrophotometer (see “Spectrophotometric quantification of RNA” below). For
small amounts of RNA, however, it may be difficult to determine amounts photometrically.
Small amounts of RNA can be quantified using the QIAxpert
®
, QIAxcel® system or Agilent®
2100 bioanalyzer, quantitative RT-PCR or fluorometric quantification.
Spectrophotometric quantification of RNA
To ensure significance, A
260
readings should be greater than 0.15. An absorbance of 1 unit
at 260 nm corresponds to 44 µg of RNA per ml (A
260
= 1 → 44 µg/ml). This relation is valid
only for measurements at a neutral pH. Therefore, if it is necessary to dilute the RNA sample,
this should be done in a buffer with neutral pH.* As discussed below (see “Purity of RNA”,
page 30), the ratio between the absorbance values at 260 and 280 nm gives an estimate of
RNA purity.
* When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For
more information, consult the appropriate safety data sheets (SDSs), available from the product supplier.
29
RNeasy Fibrous Tissue Mini Handbook 01/2018
When measuring RNA samples, be certain that cuvettes are RNase-free, especially if the RNA
is to be recovered after spectrophotometry. This can be accomplished by washing cuvettes with
0.1 M NaOH, 1 mM EDTA,* followed by washing with RNase-free water (see “Solutions”,
page 27). Use the buffer in which the RNA is diluted to zero the spectrophotometer. An
example of the calculation involved in RNA quantification is shown below:
Volume of RNA sample = 100 µl
Dilution = 10 µl RNA sample + 490 µl 10 mM Tris·Cl,* pH 7.0 (1/50 dilution)
Measure absorbance of diluted sample in a 1 ml cuvette (RNase-free)
A
260
= 0.2
Concentration of RNA sample = 44 µg/ml x A
260
x dilution factor
= 44 µg/ml x 0.2 x 50
= 440 µg/ml
Total amount = concentration x volume in milliliters
= 440 µg/ml x 0.1 ml
= 44 µg of RNA
* When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For
more information, consult the appropriate safety data sheets (SDSs), available from the product supplier.
RNeasy Fibrous Tissue Mini Handbook 01/2018
30
Purity of RNA
The ratio of the readings at 260 nm and 280 nm (A
260/A280
) provides an estimate of the
purity of RNA with respect to contaminants that absorb in the UV spectrum, such as protein.
However, the A
260/A280
ratio is influenced considerably by pH. Since water is not buffered,
the pH and the resulting A
260/A280
ratio can vary greatly. Lower pH results in a lower
A
260/A280
ratio and reduced sensitivity to protein contamination.* For accurate values, we
recommend measuring absorbance in 10 mM Tris·Cl, pH 7.5. Pure RNA has an A
260/A280
ratio of 1.9–2.1
†
in 10 mM Tris·Cl, pH 7.5. Always be sure to calibrate the
spectrophotometer with the same solution used for dilution.
For determination of RNA concentration, however, we recommend dilution of the sample in
a buffer with neutral pH since the relationship between absorbance and concentration (A
260
reading of 1 = 44 µg/ml RNA) is based on an extinction coefficient calculated for RNA at
neutral pH (see “Spectrophotometric quantification of RNA”, page 28).
To assess the purity of RNA (A
260/A280
) we recommend using the QIAxpert. The QIAxpert is
an innovative μ-volume UV/Vis spectrophotometer that overcomes the limitations of classic
spectrophotometry and purity assessment using absorbance ratios. Using reference spectra
of known contaminants, a state-of-the-art software algorithm on the QIAxpert instrument
performs a deconvolution of measured UV/Vis spectra according to the Beer Lambert law for
mixtures, stating that the absorption spectrum of a mixture is a linear combination of the
spectra of its individual constituents. This feature, known as Spectral Content Profiling (SCP),
allows dye-free and easy differentiation between DNA, RNA and other UV/Vis absorbing
contaminants in complex biological samples).
* Wilfinger, W.W., Mackey, M., and Chomczynski, P. (1997) Effect of pH and ionic strength on the
spectrophotometric assessment of nucleic acid purity. BioTechniques 22, 474.
†
Values up to 2.3 are routinely obtained for pure RNA (in 10 mM Tris·Cl, pH 7.5) with some spectrophotometers.
31
RNeasy Fibrous Tissue Mini Handbook 01/2018
DNA contamination
No currently available purification method can guarantee that RNA is completely free of
DNA, even when it is not visible on an agarose gel. While RNeasy Kits will remove the vast
majority of cellular DNA, trace amounts may still remain, depending on the amount and
nature of the sample.
For analysis of very low-abundance targets, any interference by residual DNA contamination
can be detected by performing real-time RT-PCR control experiments in which no reverse
transcriptase is added prior to the PCR step.
To prevent any interference by DNA in real-time RT-PCR applications, such as with
Rotor-Gene
®
and Applied Biosystems® instruments, we recommend designing primers that
anneal at intron splice junctions so that genomic DNA will not be amplified. QuantiNova
®
Primer Assays from QIAGEN are designed for SYBR
®
Green-based real-time RT-PCR analysis
of RNA sequences (without detection of genomic DNA) where possible (see
www.qiagen.com/GeneGlobe). For real-time RT-PCR assays where amplification of genomic
DNA cannot be avoided, we recommend using the QuantiNova Reverse Transcription Kit
(cat. no. 205411) for reverse transcription. The kit integrates fast cDNA synthesis with rapid
removal of genomic DNA contamination.
Integrity of RNA
The integrity and size distribution of total RNA purified with RNeasy Lipid Tissue Kits can be
checked by denaturing agarose gel electrophoresis and ethidium bromide staining or by
using the QIAxcel Advanced System, Agilent Tapestation or Agilent 2100 bioanalyzer. The
respective ribosomal RNAs should appear as sharp bands or peaks. The apparent ratio of
28S rRNA to 18S rRNA should be approximately 2:1. If the ribosomal bands or peaks of a
specific sample are not sharp, but appear as a smear towards smaller sized RNAs, it is likely
that the sample suffered major degradation either before or during RNA purification.
RNeasy Fibrous Tissue Mini Handbook 01/2018
32
The QIAxcel Advanced System also provides an assessment of RNA integrity using an RNA
integrity score (RIS). The RIS gives an objective quality measurement for eukaryotic RNA
samples and allows easy interpretation of sample integrity. The RIS is a value from 1 to 10
where a value of 10 indicates completely intact RNA. Similarly, the Agilent 2100
bioanalyzer offers an RNA Integrity Number (RIN) as a measure of RNA integrity. Ideally,
the RIN should be close to 10, but in many cases (particularly with tissue samples), how well
the original sample is preserved greatly influences RNA quality.
33
RNeasy Fibrous Tissue Mini Handbook 01/2018
Appendix C: DNase Digestion of RNA Eluates
Using the RNase-Free DNase Set
As an alternative to on-column DNase digestion (steps 20–23), DNase digestion of RNA
eluates can be performed using The RNase-Free DNase Set (cat. no. 79254) instead. For
samples with high DNA content, we recommend DNase digestion of RNA eluates, as it is
more efficient than on-column DNase digestion.
Important points before starting
Do not vortex the reconstituted DNase I. DNase I is especially sensitive to physical
denaturation. Mixing should only be carried out by gently inverting the tube.
Things to do before starting
Prepare DNase I stock solution before using the RNase-Free DNase Set for the first time.
Dissolve the lyophilized DNase I (1500 Kunitz units) in 550 µl of the RNase-free water
provided. To avoid loss of DNase I, do not open the vial. Inject RNase-free water into the vial
using an RNase-free needle and syringe. Mix gently by inverting the vial. Do not vortex.
For long-term storage of DNase I, remove the stock solution from the glass vial, divide it
into single-use aliquots, and store at –30 to –15°C for up to 9 months. Thawed aliquots
can be stored at 2–8°C for up to 6 weeks. Do not refreeze the aliquots after thawing.
Procedure
1. Purify RNA according to the standard protocol (page 15), but without performing the
on-column DNase digestion described in steps 20–23 (instead, perform the optional wash
with Buffer RW1 described in step 20).
RNeasy Fibrous Tissue Mini Handbook 01/2018
34
2. Mix the following in a microcentrifuge tube:
87.5 µl RNA eluate
10 µl Buffer RDD
2.5 µl DNase I stock solution
3. Make the volume up to 100 µl with RNase-free water.
The reaction volumes can be doubled if necessary (to 200 µl final volume).
4. Incubate on the benchtop (15–25°C) for 10 min.
5. Clean up the RNA using the RNeasy Mini Kit (cat. no. 74104).
35
RNeasy Fibrous Tissue Mini Handbook 01/2018
Ordering Information
Product
Contents Cat. no.
RNeasy Fibrous Tissue Mini
Kit (50)
50 RNeasy Mini Spin Columns, Collection
Tubes, Proteinase K, RNase-Free DNase I,
RNase-Free Reagents and Buffers
74704
Accessories
Allprotect Tissue Reagent
(100 ml)
For stabilization of DNA, RNA and protein
in 50 x 200 mg tissue samples: 100 ml
Allprotect Tissue Reagent, Allprotect
Reagent Pump
76405
RNAlater RNA Stabilization
Reagent (50 ml)
For stabilization of RNA in 25 x 200 mg
tissue samples: 50 ml RNAlater RNA
Stabilization Reagent
76104
RNAlater RNA Stabilization
Reagent (250 ml)
For stabilization of RNA in 125 x 200 mg
tissue samples: 250 ml RNAlater RNA
Stabilization Reagent
76106
RNAlater TissueProtect Tubes
(50 x 1.5 ml)
For stabilization of RNA in 50 x 150 mg
tissue samples: 50 screw-top tubes
containing 1.5 ml RNAlater RNA
Stabilization Reagent each
76154
RNAlater TissueProtect Tubes
(20 x 5 ml)
For stabilization of RNA in 20 x 500 mg
tissue samples: 20 screw-top tubes
containing 5 ml RNAlater RNA
Stabilization Reagent each
76163
RNeasy Fibrous Tissue Mini Handbook 01/2018
36
Product
Contents Cat. no.
TissueRuptor II Handheld rotor–stator homogenizer,
5 TissueRuptor Disposable Probes
9002755*
9002754†
9002756‡
9002757§
9002758¶
TissueLyser II Universal laboratory mixer-mill disruptor 85300
TissueRuptor Disposable
Probes (25)
25 nonsterile plastic disposable probes for
use with the TissueRuptor II
990890
TissueLyser Adapter Set 2 x
24
2 sets of Adapter Plates and 2 racks for
use with 2 ml microcentrifuge tubes on the
TissueLyser II
69982
TissueLyser Adapter Set 2 x
96
2 sets of Adapter Plates for use with
Collection Microtubes (racked) on the
TissueLyser II
69984
TissueLyser Single-Bead
Dispenser, 5 mm
For dispensing individual beads (5 mm
diameter)
69965
Stainless Steel Beads, 5 mm
(200)
Stainless Steel Beads, suitable for use with
the TissueLyser system
69989
RNase-Free DNase Set (50) 1500 Kunitz units RNase-free DNase I,
RNase-free Buffer RDD, and RNase-free
water for 50 RNA minipreps
79254
* 120 V, 60 Hz (for North America);
†
100 V, 50/60 Hz (for Japan); ‡ 235 V, 50/60 Hz (for Europe, excluding UK
and Ireland);
§
235 V, 50/60 Hz (for UK and Ireland); ¶ 235 V, 50/60 Hz (for Australia).
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.
37
RNeasy Fibrous Tissue Mini Handbook 01/2018
Limited License Agreement for RNeasy Fibrous Tiss ue Mini Kit
Use of this product signifies the agreement of any purchaser or user of the product to the following terms:
1. The product may be used solely in accordance with the protocols provided with the product and this handbook and for use with components contained in the kit
only. QIAGEN grants no license under any of its intellectual property to use or incorporate the enclosed components of this kit with any components not included
within this kit except as described in the protocols provided with the product, this handbook, and additional protocols available at www.qiagen.com. Some of
these additional protocols have been provided by QIAGEN users for QIAGEN users. These protocols have not been thoroughly tested or optimized by
QIAGEN. QIAGEN neither guarantees them nor warrants that they do not infringe the rights of third-parties.
2. Other than expressly stated licenses, QIAGEN makes no warranty that this kit and/or its use(s) do not infringe the rights of third-parties.
3. This kit and its components are licensed for one-time use and may not be reused, refurbished, or resold.
4. QIAGEN specifically disclaims any other licenses, expressed or implied other than those expressly stated.
5. The purchaser and user of the kit agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited above. QIAGEN
may enforce the prohibitions of this Limited License Agreement in any Court, and shall recover all its investigative and Court costs, including attorney fees, in any
action to enforce this Limited License Agreement or any of its intellectual property rights relating to the kit and/or its components.
For updated license terms, see
www.qiagen.com
.
Trademarks: QIAGEN®, Sample to Insight®, QIAxcel®, QIAxpert®, QIAzol®, AllProtect®, Quantiscript®, QuantiTect®, RNeasy®, Rotor-Gene®, TissueRuptor® (QIAGEN
Group); Agilent
®
(Agilent Technologies, Inc.); Applied Biosystems®, SYBR® (Thermo Fisher Scientific Inc.). Registered names, trademarks, etc. used in this document,
even when not specifically marked as such, are not to be considered unprotected by law.
“RNAlater®” is a trademark of AMBION, Inc., Austin, Texas and is covered by various U.S. and foreign patents. RNAlater may not be re-sold or used for commercial
purposes, which both require a specific license.
Jan-18 HB-0485-003 © 2018 QIAGEN, all rights reserved.
RNeasy Fibrous Tissue Mini Handbook 01/2018
38
Ordering
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