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PicoPure RNA Isolation Kit
Maximize RNA Recovery from Microscopic Samples
Isolate RNA from
Even A Single Cell
New Spin Columns and Protocol
The PicoPure RNA Isolation Kit is engineered
to recover high-quality total RNA consistently
from fewer than ten cells. The high recovery
rate attained using the Kit is valuable not only
for small samples containing picograms of RNA
but also for samples containing up to 100 µg of
RNA. Since the RNA elutes in as little as 10 µl
of buffer, it is ready for gene expression analysis
without any vacuum concentration. The Kit
is available in two sizes, enough for 40 or for
200 isolations.
Quantitative Real-Time PCR (QRT-PCR) of RNA isolated from 1 to
1000 Laser Capture Microdissected (LCM) cells. Cultured mouse
leydig (TM3) cells (ATCC) were spun down onto a microscope slide,
fixed, and dehydrated. Replicate samples of 1, 10, 100, and 1000
cells were microdissected using the PixCell IIe LCM Instrument.
RNA was isolated using the PicoPure RNA Isolation Kit, reversetranscribed and subjected to a QRT-PCR assay for GAPDH using a
LightCycler (Roche). Quantifiable message can be detected from
a single cell, and the fluorescence signal is proportional to the cell
number for all samples studied.
Get Superior
RNA Recovery
Eliminate Time-consuming
RNA Concentration
Small, valuable samples often cannot be replaced.
Performing gene expression studies on these
precious samples requires getting maximum
RNA recovery. Comparative studies show that,
with small, RNA-poor cell or tissue samples, the
PicoPure Kit dramatically outperforms other
commercial total RNA isolation kits in efficiency.
The simple isolation protocol consists of
extracting cellular RNA, then loading the extract
onto the MiraCol Purification Column to bind
the RNA. After washing away impurities, the
RNA elutes in only 10 µl of buffer, ready for use
without vacuum concentration or transfer into a
new reaction tube.
Use High-yield
Purification Columns
Why Use the PicoPure Kit?
• Consistent, efficient RNA Recovery
• Low-volume elution
• Compatible with several tube sizes
• Column lid to prevent contamination
RNA recovery efficiency comparison among commercial RNA
isolation kits. Using the PicoPure Kit and other leading products
for purifying small RNA samples, 100 ng of Cy3-labeled RNA was
bound to columns, washed, and eluted following the corresponding
manufacturer’s protocol. Values represent average percent recovery
of RNA relative to the PicoPure Kit, of five replicates per kit, quantified
using fluorescence detection.
The PicoPure RNA Isolation Kit features
unique, high-recovery MiraCol Purification
Columns, engineered to maximize recovery
from microscopic samples. The columns have
convenient integrated lids to help ensure that
samples remain RNase free. In addition, the Kit
comes complete with all necessary buffers, an
easy-to-follow User Guide, optimized protocols
for handling Laser Capture Microdissected
(LCM) samples, and protocols for isolating RNA
from larger samples.
Deliver RNA Ready for Amplification
and Microarray Analysis
Maintain High
RNA Quality
RNA isolated with the PicoPure RNA Kit
is ready, without vacuum concentration, for
amplification with the RiboAmp RNA
The PicoPure RNA isolation process ensures
high-quality RNA. Total cellular RNA prepared
from LCM samples remains intact after isolation,
increasing confidence in subsequent molecular
analysis.
Amplification Kit. The two kits together
provide highly reproducible microarray results.
B
A
C
The PicoPure Kit maintains RNA
integrity. Total RNA from 15,000
mouse kidney cells was isolated from
two samples with the PicoPure RNA
Isolation Kit. RNA samples were run
using an Agilent Bioanalyzer 2100.
Lane 1: Total RNA from non-LCM cells.
Lane 2: Total RNA from LCM-captured
cells. Comparable 18S and 28S
ribosomal RNA band intensities are
observed between RNA isolated from
both the intact tissue and the LCM
sample, demonstrating high quality of
RNA obtained.
Reproducible amplification and microarray results using PicoPure-isolated RNA. Panel A. Total RNA was isolated from six independent
LCM samples using the PicoPure RNA Isolation Kit. RNA from each capture was amplified two rounds using the RiboAmp RNA Amplification
Kit. Amplified aRNA was run on an agarose gel. M: marker. Lane 1: positive amplification control. Lane 2: negative control. Lane 3: kidney duct
1. Lane 4: kidney duct 2. Lane 5: mouse kidney glomerulus. Lane 6: whole kidney. Lane 7: SKBR3 cell line (250 captured cells). Lane 8: SKBR3
cell line (500 captured cells). Panels B-C. RNA from 1500 mouse kidney duct cells was isolated using the PicoPure RNA Isolation Kit, amplified
two rounds using the RiboAmp Kit, labeled with Cy5-dUTP, and hybridized onto 9000- element mouse cDNA arrays. One region of two arrays
illustrates high correlation between gene expression patterns (R = 0.979).
Ordering Information
Retain Low-abundance mRNA
The PicoPure RNA Isolation Kit preserves
mRNA in all abundance classes through
the isolation process, minimizing the loss of
important genes of interest. Low, medium, and
high-abundance genes are consistently recovered
and available for expression analysis.
RT-PCR of low, medium, and high-abundance genes in RNA
isolated using the PicoPure Kit. RT-PCR was performed on RNA
from 500 cells captured from mouse liver and kidney. Equal
quantities of cDNA were analyzed with three mouse primer
sets: Elongation Factor 1-a (EF-1a, high-abundance gene, ~3000
copies/cell, 187 bp), Glyceraldehyde-3-phosphate Dehydrogenase
(GAPDH, medium- abundance gene, 300-3000 copies/cell, 357
bp), and Protein Phosphatase 1 (PP1, low-abundance gene, <300
copies/cell, 498 bp). Samples were run on an acrylamide gel.
M: molecular weight markers. Lane 1: kidney EF-1a. Lane 2: liver
EF-1a. Lane 3: kidney GAPDH. Lane 4: liver GAPDH. Lane 5: kidney
PP1. Lane 6: liver PP1. Lane 7 liver EF-1a no RT control. Lane 8: no RT
template control.
Catalog #
KIT0204
Description
PicoPureTM RNA Isolation Kit, enough for
40 RNA isolations
KIT0214
PicoPureTM RNA Isolation Kit, enough for
200 RNA isolations
Related Products
Catalog #
KIT0401
Description
HistoGeneTM LCM Frozen Section Staining
Kit, enough for 72 tissue sections
KIT0201
RiboAmpTM RNA Amplification Kit, enough
for 10 amplifications
KIT0215
RiboAmpTM HS RNA Amplification Kit–Bulk
Pack, enough for 20 amplifications
KIT0103
PicoPureTM DNA Extraction Kit–1500 µl
Buffer (150 CapSure HS/ 30 CapSure Macro
LCM Cap Extractions)
LCM0213
CapSure HS LCM Starter Pack with
Alignment Tray and Incubation Block, 24
caps/box
LCM1105
PixCell® IIe Laser Capture Microdissection
Instrument with PixCell IIe Image Archiving
Workstation, 110V
RiboAmp, PicoPure, HistoGene, PixCell, CapSure and ExtracSure are
trademarks owned by Arcturus. All other trademarks shown are the
property of their respective owners. Polymerase Chain Reaction (PCR)
is a patented process owned by Hoffman-La Roche AG.
For research use only.
Arcturus
400 Logue Avenue
Mountain View, CA
USA 94043
www.arctur.com
PN 13662-00 Rev A
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650.962.3039 fax
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User Guide
PicoPure

RNA Isolation Kit
Catalog # KIT0202, KIT0204
Version D
For Research Use Only
PicoPure RNA Isolation Kit
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Table of Contents
I. Introduction
A.
B.
C.
D.
E.
F.
Background
Performance Specifications
Quality Control
Storage and Stability
Material Safety and Data Sheets
Related Products Available from Arcturus
3
3
4
4
4
5
II. Kit Components
A. Reagents and Supplies Provided
7
III. Preliminary Steps
A.
B.
C.
D.
E.
Recommendations for RNase-free Technique
Recommendations for Storing RNA
Additional Lab Equipment and Materials Required
DNase Treatment of Samples
Nucleic Acid Elution Using Spin Columns
8
8
9
9
9
IV. Protocol
A. Overview
B. Protocol for Use with CapSure HS LCM Caps
C. Protocol for Use with CapSure Macro LCM Caps
10
11
14
V. Troubleshooting
A. Isolated RNA is of Poor Quality
B. RNA Yield is Low
17
18
VI. Appendices
A. DNase Treatment
B. RNA Extraction from Cell Pellets
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1
PicoPure RNA Isolation Kit
Legal Notices
Copyright © 2004 Arcturus Bioscience, Inc. All Rights Reserved.
PixCell ®, CapSure ® and RiboAmp ® are registered trademarks, and
ExtracSure, HistoGene, PicoPure, AutoPix and Paradise are
trademarks of Arcturus Bioscience, Inc. Other trademarks used in this
manual are the properties of their respective owners. The PCR process is
covered by patents owned by Hoffman-La Roche Inc., and F. Hoffman-La
Roche Ltd.
Warranty
Arcturus Bioscience, Inc. warrants that the products described in this
manual meet the performance standards described in literature published
by the company. If a product fails to meet these performance standards,
Arcturus will replace the product or issue credit for the full purchase price,
including delivery charges. Arcturus provides no other warranties of any
kind, expressed or implied. Arcturus’ warranty liability shall not exceed
the purchase price of the product and shall not extend to direct, indirect,
consequential or incidental damages arising from the use, results of use, or
improper use of its products. The PicoPure RNA Isolation Kit is intended
for research purposes only and is not intended for diagnostic or other
purposes.
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Introduction
I. Introduction
A. Background
The PicoPure RNA Isolation Kit enables researchers to recover
total cellular RNA from pico-scale samples. The PicoPure RNA
Isolation Kit is optimized for use with cells acquired using Laser
Capture Microdissection (LCM) on CapSureHS LCM Caps
and CapSure Macro LCM Caps but can be used with various
cell samples to isolate total cellular RNA. Researchers can obtain
high recoveries of total cellular RNA from as little as a single cell to
samples with up to 100 µg of RNA. Total cellular RNA isolated
using the PicoPure RNA Isolation Kit produces RNA in a small
volume of low ionic strength buffer, ready for use in downstream
applications including reverse transcription for Q-PCR,
production of labeled cDNA, or linear amplification using the
RiboAmp RNA Amplification Kit.
The PicoPure RNA Isolation Kit contains RNA extraction and
purification reagents, RNA purification columns, and this User
Guide.
B. Performance Specifications
RNA isolated using the PicoPure Kit is intact and ready for
downstream applications when starting with samples with high
RNA quality. RT-PCR of GAPDH from isolated total RNA
from 100 LCM captured cells prepared using the HistoGene
LCM Frozen Section Staining Kit produces visible product upon
gel electrophoresis separation and staining.
Researchers can complete total cellular RNA isolations from cells
captured on CapSure HS LCM Caps or CapSure Macro LCM
Caps in less than an hour. RNA extraction from LCM caps requires
30 minutes of incubation time, and the RNA purification process
takes less than 20 minutes.
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PicoPure RNA Isolation Kit
C. Quality Control
Arcturus performs functional testing on the PicoPure RNA
Isolation Kit using all kit components and the protocol described
in this user guide. Using the kit, Quality Control Specialists
isolate RNA from 100 cells acquired by LCM and analyze it by
RT-PCR using primers to generate amplicons of a specific gene or
gene fragment. The reaction product is separated by
electrophoresis and stained. A single amplicon band of the correct
size is visualized.
RNA purification columns are tested by lot to confirm the absence
of nucleic acids and nuclease activity. Column nucleic acid
binding and recovery performance must meet quality standards.
A Quality Control Certificate included with each shipment
describes the tests performed and includes individual lot testing
data.
D. Storage and Stability
Store the PicoPure RNA Isolation Kit at room temperature.
Properly stored kits are stable until the expiration date indicated
on the package.
E. Material Safety and Data Sheets
Material Safety and Data Sheets (MSDS) for kit chemical
components are available from Arcturus Technical Services. Call
888.446.7911 or 650.962.3020, send e-mail to
[email protected], or download the file from
www.arctur.com.
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Introduction
F. Related Products from Arcturus
CapSure HS LCM Caps
CapSure HS LCM Caps enable the highest specificity cell capture
from LCM. CapSure HS LCM Caps come with PrepStrip
Tissue Preparation Strips and ExtracSure Sample Extraction
Devices.
CapSure Macro LCM Caps
CapSure Macro LCM Caps enable large quantity LCM captures.
Each box of caps also contains PrepStrip Tissue Preparation Strips.
HistoGene
 LCM Frozen Section Staining Kit
The HistoGene LCM Frozen Section Staining Kit simplifies tissue
staining and dehydration while maintaining RNA integrity. The
HistoGene Kit uses an optimized tissue section processing system
that includes reagents and protocols for preserving tissue and
cellular RNA to maximize quality and yield of RNA from cells
acquired by LCM.
HistoGene LCM Immunofluorescence Staining Kit
The HistoGene LCM Immunofluorescence Staining Kit is the
only kit designed to enable retrieval of high-quality RNA from
immunofluorescently stained frozen tissue. It enables convenient
and reliable staining, dehydration and LCM of tissue sections
with protocols streamlined and optimized both for optimal LCM
captures and maintaining RNA quality for downstream
applications that require intact RNA, like microarray analysis and
RT-PCR.
RiboAmp RNA Amplification Kit
The RiboAmp RNA Amplification Kit enables the production
of microgram quantities of antisense RNA (aRNA) from
nanogram quantities of total cellular RNA. Amplified RNA
produced using the kit is suitable for labeling and use for probing
expression microarrays. The kit achieves amplifications of up to
1000-fold in one round of amplification, and amplifications of
up to 1,000,000-fold in two rounds. The RiboAmp Kit comes
with all necessary enzymes, reagents, and nucleic acid purification
devices needed to complete the included amplification protocol.
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PicoPure RNA Isolation Kit
RiboAmp HS RNA Amplification Kit
The RiboAmp HS RNA Amplification Kit starts with picogram
total cellular RNA input and enables the production of microgram
quantities of antisense RNA (aRNA). The kit provides the greatest
level of sensitivity in starting RNA quantities to produce enough
RNA suitable for labeling and hybridizing onto expression
microarrays. The RiboAmp HS Kit come with all necessary
enzymes, reagents, and MiraCol Purification Columns needed
to complete the included amplification protocol.
RiboAmp OA RNA Amplification Kit
The RiboAmp OA RNA Amplification Kit enables the
production of microgram quantities of antisense RNA (aRNA)
from nanogram quantities of total cellular RNA. This specially
formulated kit conveniently prepares small samples for direct
labeling to hybridize onto oligonucleotide arrays requiring labeled
aRNA, such as GeneChip® Probe Arrays (Affymetrix). Also, the
Kit may be used for preparation of labeled aRNA to hybridize
onto cDNA platforms while eliminating the need for postamplification labeling. The RiboAmp OA Kit is coupled with
commercially available transcript labeling systems that incorporate
either biotin or fluorescent labels directly into aRNA.
 Reagent System
Paradise
The Paradise Reagent System is the only reagent system desinged
to enable gene expression studies using formalin-fixed paraffinembedded (FFPE) tissue samples. Components include sample
preparation and staining reagents, RNA extraction and isolation
reagents, RNA amplification reagents and a comprehensive user
guide.
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Kit Components
II. Kit Components
A. Reagents and Supplies Provided
The PicoPure RNA Isolation Kit comes with the following items:
Item
Conditioning Buffer
Extraction Buffer
70% Ethanol
Wash Buffer 1
Wash Buffer 2
Elution Buffer
RNA purification columns with
collection tubes
Microcentrifuge tubes
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Vial Name
CB
XB
EtOH
W1
W2
EB
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PicoPure RNA Isolation Kit
III. Preliminary Steps
A. Recommendations for RNase-free Technique
RNase contamination will cause experimental failure. Minimize
RNase contamination by adhering to the following
recommendations throughout your experiment:
•
Always handle RNA in a manner that avoids introduction of
RNases.
•
Wear disposable gloves and change them frequently to prevent
the introduction of RNases from skin surfaces.
•
After putting on gloves, avoid touching surfaces that may
introduce RNases onto glove surfaces.
•
Do not use reagents not supplied in the PicoPure RNA
Isolation Kit. Substitution of reagents or kit components may
adversely affect yields or introduce RNases.
•
Use only new plasticware that is certified nucleic acid-free.
•
Use only new, sterile, RNase-free pipette tips and microcentrifuge tubes.
•
Clean work surfaces with commercially available RNase
decontamination solutions prior to performing reactions.
B. Recommendations for Storing RNA
Begin the PicoPure RNA Isolation Kit protocol immediately
following acquisition of cells by LCM. The Extraction Buffer
(XB) stabilizes RNA. Cell extracts resulting from completion of
Part I of the protocol may be stored at –80°C.
Following protocol completion, use isolated RNA immediately
for amplification, or store at –80°C for up to 6 months.
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Preliminary Steps
C. Additional Lab Equipment and Materials Required
Ensure that you have ready access to the following laboratory
equipment and materials before you begin. These items are not
included in the PicoPure RNA Isolation Kit:
1. Equipment
‰ Microcentrifuge (Eppendorf 5415D or similar)
‰ 2–20 µl pipettor
‰ 20–200 µl pipettor
‰ Incubation oven
2. Materials
‰ Nuclease-free pipette tips
‰ 0.5 ml microcentrifuge tubes (Applied BioSystems Catalog
#N8010611)
‰ 2 ml lidless tubes (PGC Scientific, Catalog #16-8101-06)
3. Reagents (optional)
‰ DNase, RNase-free (see Appendix)
D. DNase Treatment of Samples
The enhanced sensitivity of the PicoPure RNA Isolation Kit is
made possible by the use of proprietary components. Some of
these components may interfere with techniques to assess RNA
quality such as gel electrophoresis or using the Agilent 2100
BioAnalyzer. In order to prevent these artifacts as well as genomic
DNA contamination, we strongly recommend DNase treatment.
In addition, if performing reverse transcription or amplification
using RiboAmp RNA amplification Kits after RNA isolation,
DNase treatment is recommended. Please see Appendix A for a
detailed protocol.
E. Nucleic Acid Elution Using Spin Columns
Spin columns and 0.5 ml microcentrifuge tubes are provided for
nucleic acid elution. Improper orientation of tubes, with caps
open during centrifugation, may result in cap breakage or sample
loss. To correctly use the column-tube assembly, insert a spin
column into the 0.5 ml tube, aligning the two cap hinges as
illustrated. Load Elution Buffer onto the column and incubate
as directed. Place the column-tube assembly into a 2 ml lidless
support tube (PGC Scientific, Catalog #16-8101-06) in the
centrifuge rotor. Skip one rotor position between assemblies, and
position assemblies with the 0.5 ml tube cap trailing the tube
during centrifugation as shown. (Check for a mark on the
centrifuge indicating rotation direction.) Centrifuge as directed
in the protocol.
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PicoPure RNA Isolation Kit
IV. Protocol
A. Overview
The flow chart illustrates the PicoPure RNA Isolation Kit
procedure: (a) Extract RNA from a CapSure HS LCM Cap or a
CapSure Macro LCM Cap. (b) Load cell extract onto a preconditioned purification column. (c) Spin the extract through
the column to capture RNA on the purification column
membrane. (d) Wash the column twice with wash buffer, and (e)
elute the RNA in low ionic strength buffer. The entire process,
including incubations, can be completed in less than an hour,
and the isolated total cellular RNA is ready for use in downstream
applications.
PicoPure is capable of isolating extremely small amounts of RNA.
It is important not to introduce nucleic acid contamination.
If you used CapSure HS LCM Caps to capture cells for RNA
isolation, use the following protocol presented under heading B.
If you used Capsure Macro LCM Caps to capture cells, use the
protocol presented under C on page 14. For RNA isolation of
cell pellets, use the protocol presented in Appendix B on
page 20.
(a)
(b)
(c)
(d)
(e)
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Protocol
B. Protocol for Use with CapSure HS LCM Caps
1.
RNA Extraction
a.
Dispense Extraction Buffer (XB) and incubate:
i.
Capture cells and assemble the CapSure HS Cap with
the ExtracSure Extraction Device. Refer to the CapSure
HS Caps User Guide for complete instructions.
ii.
Place the CapSure–ExtracSure assembly in a CapSure
HS Alignment Tray and pipette 10 µL Extraction
Buffer (XB) into the buffer well.
Prior to use, mix Extraction Buffer
(XB) thoroughly. Extraction Buffer
(XB) may form precipitate upon
storage. Dissolve precipitate prior to
use by mixing thoroughly. If necessary,
warm the XB vial to redissolve
Extraction Buffer prior to use.
Pipettor Tip
ExtracSure Sample
Extraction Device
iii. Place a new 0.5 mL microcentrifuge tube (Applied
BioSystems Catalog # N8010611) onto the CapSure–
ExtracSure assembly. Cover with Incubation Block
preheated to 42°C.
Heating Block
iv. Incubate assembly for 30 minutes at 42°C.
Alignment Tray
b.
Centrifuge the microcentrifuge tube with the CapSure–
ExtracSure assembly at 800 x g for two minutes to collect
cell extract into the microcentrifuge tube.
c.
After centrifugation, the microcentrifuge tube contains the
cell extract required to complete the protocol. Remove the
microcentrifuge tube from the CapSure–ExtracSure
assembly and save the microcentrifuge tube with the cell
extract in it.
d.
Proceed with RNA isolation protocol or freeze cell extract at
–80°C.
CapSure–ExtracSure
Assembly with
Microcentrifuge
Tube
The CapSure HS LCM Caps and
the ExtracSure Device are compatible
with 0.5 ml thin-walled reaction
tubes from Applied BioSystems. These
reaction tubes should be used for RNA
extraction.
It is okay to stop at this point in the protocol.
2. RNA Isolation
a.
Pre-condition the RNA Purification Column:
i.
Pipette 250 µL Conditioning Buffer (CB) onto the
purification column filter membrane.
ii.
Incubate the RNA Purification Column with Conditioning Buffer for 5 minutes at room temperature.
iii. Centrifuge the purification column in the provided
collection tube at 16,000 x g for one minute.
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PicoPure RNA Isolation Kit
b.
Pipette 10 µL of 70% Ethanol (EtOH) into the cell extract
from Part 1 (RNA Extraction). Mix well by pipetting up
and down. DO NOT CENTRIFUGE.
c.
Pipette the cell extract and EtOH mixture into the preconditioned purification column. The cell extract and EtOH
will have a combined volume of approximately 20 µL.
d.
To bind RNA, centrifuge for 2 minutes at 100 x g,
immediately followed by a centrifugation at 16,000 x g for
30 seconds to remove flowthrough.
e.
Pipette 100 µL Wash Buffer 1 (W1) into the purification
column and centrifuge for one minute at 8,000 x g.
Optional: DNA may be removed by DNase treatment from the
preparation at this point. See Appendix A for a detailed
protocol.
f.
Pipette 100 µL Wash Buffer 2 (W2) into the purification
column and centrifuge for one minute at 8,000 x g.
g.
Pipette another 100 µL Wash Buffer 2 (W2) into the purification column and centrifuge for two minutes at 16,000 x
g. Check the purification column for any residual wash
buffer. If wash buffer remains re-centrifuge at 16,000 x g
for one minute.
h.
Transfer the purification column to a new 0.5 mL
microcentrifuge tube provided in the kit.
i.
Pipette Elution Buffer (EB) directly onto the membrane of
the purification column (Gently touch the tip of the pipette
to the surface of the membrane while dispensing the elution
buffer to ensure maximum absorption of EB into the
membrane). Use the Elution Volume Guide table to select
the correct volume of Elution Buffer to use in this step.
Flowthrough waste following
centrifugation is usually present as only
a small volume, and therefore it is not
necessary to discard the flowthrough
waste after every centrifugation step.
Make sure that the accumulated
flowthrough waste does not make
contact with the purification column.
Flowthrough waste should be
discarded when the waste fluid level
approaches the surface of the
purification column.
DNase treatment is recommended if
performing reverse transcription or
amplification with the RiboAmp RNA
Amplification Kit after RNA isolation.
Remove all traces of wash buffer prior
to transferring purification column to
the new microcentrifuge tube. To
remove wash buffer, discard flowthrough waste and recentrifuge
the column for one minute at
16,000 x g.
Elution Volume Guide
Recommended Volume
Maximum Volume
11 µL
30 µL
The eluted RNA can be used directly in the RiboAmp RNA
Amplification Kit or in reverse transcription protocols. For
samples eluted in the maximum volume, the eluted RNA may
be concentrated in a vacuum centrifuge to reduce the volume
to 10 µL.
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Protocol
j.
Incubate the column for one minute at room temperature.
k.
Centrifuge the column for one minute at 1,000 x g to
distribute EB in the column, and then spin for one minute
at 16,000 x g to elute RNA. The isolated RNA is now ready
for use in downstream applications such as reverse
transcription or amplification with the RiboAmp RNA
Amplification Kit. The entire sample may be used
immediately or stored at –80°C until use.
To avoid potential breakage of the
microcentrifuge tube cap during
centrifugation, insert the purification
column/ 0.5 mL tube assembly into a
lidless 1.7/2.0 mL tube. Insert this
assembly into adjacent rotor holes as
illustrated. Rest the tube cap against
the tube immediately clockwise to it.
Place an empty, lidless 1.7/2.0 mL
tube into the rotor hole adjacent in
the clockwise direction to the last
assembly.
Quantitation of isolated RNA through
UV spectrophotometry or measurement by fluorescence based methods
may not be possible for samples
containing less than 1 µg of total RNA.
Measurements will be affected by
components in the eluted sample,
which cause an overestimation of the
total RNA content.
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PicoPure RNA Isolation Kit
C. Protocol for Use with CapSure Macro LCM Caps
1.
RNA Extraction
a.
Dispense Extraction Buffer (XB) and incubate:
i.
Pipette 50 µL Extraction Buffer (XB) into a 0.5 mL
microcentrifuge tube (Applied BioSystems Catalog #
N8010611).
ii.
Insert CapSure Macro LCM Cap onto the
microcentrifuge tube using an LCM Cap Insertion Tool.
Prior to use, mix Extraction Buffer
(XB) thoroughly. Extraction Buffer
(XB) may form precipitate upon
storage. Dissolve precipitate prior to
use by mixing thoroughly. If necessary,
warm the XB vial to redissolve
Extraction Buffer prior to use.
iii. Invert the CapSure Cap–microcentrifuge tube assembly.
Tap the microcentrifuge tube to ensure all Extraction
Buffer (XB) is covering the CapSure Macro LCM Cap.
iv. Incubate assembly for 30 minutes at 42°C.
b.
Centrifuge assembly at 800 x g for two minutes to collect
cell extract into the microcentrifuge tube.
c.
After centrifugation, the microcentrifuge tube contains the
cell extract required to complete the protocol. Remove the
CapSure Macro LCM Cap and save the microcentrifuge
tube with the cell extract in it.
d.
Proceed with RNA isolation protocol or freeze cell extract at
–80°C.
It is okay to stop at this point in the protocol.
2.
RNA Isolation
a.
Pre-condition the RNA Purification Column:
i.
Pipette 250 µL Conditioning Buffer (CB) onto the
purification column filter membrane.
ii.
Incubate the RNA Purification Column with
Conditioning Buffer for 5 minutes at room temperature.
iii. Centrifuge the purification column in the provided
collection tube at 16,000 x g for one minute.
b.
Pipette 50 µL of 70% Ethanol (EtOH) into the cell extract
from Part 1 (RNA Extraction). Mix well by pipetting up
and down. DO NOT CENTRIFUGE.
c.
Pipette the cell extract and EtOH mixture into the preconditioned purification column. The cell extract and EtOH
will have a combined volume of approximately 100 µL.
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Flowthrough waste following
centrifugation is usually present as only
a small volume, and therefore it is not
necessary to discard the flowthrough
waste after every centrifugation step.
Make sure that the accumulated flowthrough waste does not make contact
with the purification column. Flowthrough waste should be discarded
when the waste fluid level approaches
the surface of the purification column.
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Protocol
d.
To bind RNA to the column, centrifuge for 2 minutes at
100 x g, immediately followed by a centrifugation at
16,000 x g for 30 seconds to remove flowthrough.
e.
Pipette 100 µL Wash Buffer (W1) into the purification
column and centrifuge for one minute at 8,000 x g.
DNase treatment is recommended if
performing reverse transcription or
amplification with the RiboAmp RNA
Amplification Kit after RNA isolation.
Optional: DNA may be removed by DNase treatment from
the preparation at this point. See Appendix A for a detailed
protocol.
f.
Pipette 100 µL Wash Buffer 2 (W2) into the purification
column and centrifuge for one minute at 8,000 x g.
g.
Pipette another 100 µL Wash Buffer (W2) into the purification column and centrifuge for two minutes at 16,000 x
g. Check the purification column for any residual wash
buffer. If wash buffer remains re-centrifuge at 16,000 x g
for one minute.
h.
Transfer the purification column to a new 0.5 mL
microcentrifuge tube provided in the kit.
i.
Pipette Elution Buffer (EB) directly onto the membrane of
the purification column (Gently touch the tip of the pipette
to the surface of the membrane while dispensing the elution
buffer to ensure maximum absorption of EB into the
membrane). Use the Elution Volume Guide table to select
the correct volume of the Elution Buffer to use in this step.
Remove all traces of wash buffer prior
to transferring purification column to
the new microcentrifuge tube. To
remove wash buffer, discard flowthrough waste and recentrifuge
the column for one minute at
16,000 x g.
Elution Volume Guide
Recommended Volume
Maximum Volume
11 µL
30 µL
The eluted RNA can be used directly in the RiboAmp RNA
Amplification Kit or in reverse transcription protocols. For
samples eluted in the maximum volume, the eluted RNA may
be concentrated in a vacuum centrifuge to reduce the volume
to 10 µL.
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PicoPure RNA Isolation Kit
j.
Incubate the purification column for one minute at room
temperature.
k.
Centrifuge the column for one minute at 1,000 x g to
distribute EB in the column, then for one minute at 16,000
x g to elute RNA. The isolated RNA is now ready for use in
downstream applications such as reverse transcription or
amplification with the RiboAmp Kit. The entire sample
may be used immediately or stored at –80°C until use.
To avoid potential breakage of the
microcentrifuge tube cap during
centrifugation, insert the purification
column/ 0.5 mL tube assembly into a
lidless 1.7/2.0 mL tube. Insert this
assembly into adjacent rotor holes as
illustrated. Rest the tube cap against
the tube immediately clockwise to it.
Place an empty, lidless 1.7/2.0 mL
tube into the rotor hole adjacent in
the clockwise direction to the last
assembly.
Quantitation of isolated RNA through
UV spectrophotometry or measurement by fluorescence based methods
may not be possible for samples
containing less than 1 µg of total RNA.
Measurements will be affected by
components in the eluted sample,
which cause an overestimation of the
total RNA content.
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V. Troubleshooting
A. Isolated RNA is of Poor Quality
1.
Verify quality of source tissue of LCM cells. The greatest
factor affecting the quality of isolated RNA is the integrity
of the RNA in the original tissue sample. RNA degradation
due to RNase activity occurs rapidly, especially upon tissue
removal such as through biopsy and needle aspiration. Tissue
should be embedded and frozen immediately upon removal
to reduce RNase activity. For suggestions on verifying quality,
please call Arcturus Technical Support.
2.
Use the HistoGene LCM Frozen Section Staining Kit to
prepare slides for LCM. Specialized staining protocols and
reagents are required for optimal RNA preservation in LCM
samples. Arcturus has developed and validated the
HistoGene LCM Frozen Section Staining Kit for preparing
and staining tissues for LCM while maintaining RNA
integrity.
3.
Perform LCM immediately after preparing LCM slides.
LCM sample slides are dehydrated in the final step of
preparation, so RNase activity is minimized. However, the
risk of moisture and RNases entering the sample following
preparation increases with the interval of time between slide
preparation and RNA isolation.
4.
Pipette PicoPure Extraction Buffer (XB) onto cells acquired
by LCM immediately after cell capture. Extraction Buffer
(XB) stabilizes RNA by denaturing nucleases. Complete
Part 1 (RNA Extraction) of the PicoPure RNA Isolation Kit
Protocol without stopping. The cell extract resulting from
the completion of Part I (RNA Extraction) may be stored in
Extraction Buffer (XB) at –80°C.
5.
Use only frozen or alcohol-fixed tissue or cytosmeared cells.
Using tissue or cell samples that have been subjected to
cross-linking fixative such as formalin or paraformaldehyde
diminishes RNA quality. PicoPure Extraction Buffer (XB) is
not suitable for extraction of RNA from cross-linked tissue.
For isolation of RNA from formalin-fixed, paraffinembedded (FFPE) samples, use the Paradise Reagent System.
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PicoPure RNA Isolation Kit
Troubleshooting
B. RNA Yield is Low
1. RNA integrity has been compromised. Verify quality of initial
tissue sample or LCM slide (see A.1). Poor quality RNA may
not bind effectively to the purification column membrane,
decreasing overall RNA yield.
2. Buffer concentrations in extraction mixtures are incorrect
due to inadequate mixing with 70% Ethanol. Ensure all
buffers are completely mixed and all solids are dissolved
prior to use.
3. Elution Buffer (EB) concentration is incorrect due to
contamination with Wash Buffer 2 (W2). Ensure that all
Wash Buffer 2 has been removed by centrifugation before
proceeding to add Elution Buffer. Residual Wash Buffer 2
(W2) on the purification column filter membrane will alter
the concentration of Elution Buffer (EB), resulting in poor
RNA elution. If any Wash Buffer 2 (W2) remains in or on
the purification column, recentrifuge it to remove the residual
buffer before proceeding to elution.
4. Extraction step incubation was too short. Incubate the LCM
sample in Extraction Buffer (XB) for a full 30 minutes at
42°C. Complete cell extraction from fixed, dehydrated
samples requires this validated incubation condition.
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VI. Appendix
A. DNase Treatment
DNase treatment may be performed directly within the
purification column when downstream applications require
removal of genomic DNA to reduce risk of DNA interference..
The following protocol utilizing the RNase-Free DNase Set
(Qiagen, catalog#79254) may be used.
Protocol is applied during Part 2, RNA Isolation protocol between
Steps e and f.
1. Pipette 5 µL DNase I Stock Solution to 35 µL Buffer RDD
(provided with RNase-Free DNase Set). Mix by gently
inverting. For isolations from larger samples such as cell
pellets (see Appendix B), pipette 10 µl DNase I stock solution
to 30 µL Buffer RDD.
2. Pipette the 40 µL DNase incubation mix directly into the
purification column membrane. Incubate at room
temperature for 15 minutes.
3. Pipette 40 µL PicoPure RNA Kit Wash Buffer 1 (W1) into
the purification column membrane. Centrifuge at 8000 x
g for 15 seconds.
4. Proceed with RNA Isolation protocol Step f of Section IV,
B2 or Section IV C2.
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PicoPure RNA Isolation Kit
B. RNA Extraction from Cell Pellets
The PicoPure RNA Isolation Kit may be used for non-LCM
samples such as cell sample cultures in suspension1. The following
is a suggested protocol for the treatment of cell culture cells in
suspension. Adherent cells need to be detached from the cell
culture flask or disk prior to preceeding.
The protocol replaces Part 1:RNA Extraction of either protocol
in Chapter IV.
1. Pellet cells in a microcentrifuge tube by centrifuging at 3,000
x g for 10 minutes.
2. Properly dispose of the supernatant.
3. Resuspend the cell pellet in one mL of cell suspension media
(0.9 mL of 1 x PBS/10%BSA; 0.1 mL of 0.5 M EDTA).
DO NOT VORTEX.
4. Centrifuge the cell suspension at 3,000 x g for five minutes.
5. Properly dispose of supernatant.
6. Extract the cells with 100 µL of Extraction Buffer (XB).
Resuspend the cell pellet gently by pipetting. DO NOT
VORTEX.
7. Incubate at 42° for 30 minutes.
8. Centrifuge the sample at 3,000 x g for two minutes.
9. Pipette the supernatant containing the extracted RNA into
a new microcentrifuge tube, avoiding pick-up of pelleted
material.
10. Proceed with RNA Isolation or freeze at –70° C.
11. Proceed with the RNA Isolation protocol in sections IV:B or
IV:C with the following modification to step b:
Pipette 100 µL of 70% Ethanol (EtOH) to the cell extract
(or equal volume to the cell extract). Mix well by pipetting
up and down. DO NOT CENTRIFUGE. The combined
volume will be approximately 200 µL.
1
The PicoPure RNA Isolation Kit is not recommended for RNA isolation
from whole blood, plant, or fungal samples. The addition of whole tissues
and cells (e.g. whole blood samples) directly onto the purification columns
is not recommended.
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400 Logue Avenue
Mountain View, CA
USA 94043
www.arctur.com
888.446.7911
650.962.3020 tel
650.962.3039 fax
[email protected]
Arcturus GmbH
Germany
[49] (0) 61 05-40 88 0 tel
[49] (0) 61 05-40 88 40 fax
[email protected]
Arcturus Ltd.
United Kingdom
[44] (158) 246-9010 tel
[44] (158) 246-7988 fax
[email protected]
p/n 12682-00