Ovation® One-Direct System

38 Pages·2014·0.31 MB·English

USER GUIDE
Ovation® One-Direct System
PART NO. 3500
Patents, Licensing and Trademarks
©2009–2014 NuGEN Technologies, Inc. All rights reserved. The Encore®, Ovation® and
Applause® families of products and methods of their use are covered by several issued U.S. and
International patents and pending applications (www.nugen.com). NuGEN, Ovation, SPIA, Ribo-
SPIA, Applause, Encore, Prelude, Mondrian and Imagine More From Less are trademarks or
registered trademarks of NuGEN Technologies, Inc. Other marks appearing in these materials are
marks of their respective owners.
The purchase of this product conveys to the buyer the limited, non-exclusive, non-transferable
right (without the right to modify, reverse engineer, resell, repackage or further sublicense) under
these patent applications and any patents issuing from these patent applications to use this prod-
uct and methods, accompanying this user guide, for research and development purposes solely
in accordance with the intended use described and the written instructions provided in this user
guide. No license to make or sell products by use of this product is granted to the buyer whether
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does not include or carry any right or license to use, develop or otherwise exploit this product
commercially and no rights are conveyed to the buyer to use the product or components of the
product for purposes including commercial services or clinical diagnostics.
For information on purchasing a license to the NuGEN patents for uses other than in conjunction
with this product or to use this product for purposes other than research, please contact NuGEN
Technologies, Inc., 201 Industrial Road, Suite 310, San Carlos, CA 94070. Phone [phone] or
650-590-3600; FAX [phone] or 650-590-3630.
Warranty
NuGEN warrants that this product meets the performance standards described in the Company’s
product and technical literature for a period of six months from the date of purchase, provided
that the product is handled and stored according to published instructions, and that the product
is not altered or misused. If the product fails to meet these performance standards, NuGEN will
replace the product free of charge or issue a credit for the purchase price. NuGEN’s liability under
this warranty shall not exceed the purchase price of the product. NuGEN shall assume no liability
for direct, indirect, consequential or incidental damages arising from the use, results of use or
inability to use its products. NuGEN reserves the right to change, alter or modify any product to
enhance its performance and design.
NuGEN’s products are developed, designed and sold FOR RESEARCH USE ONLY. This product is
not to be used for diagnostic or therapeutic purposes, nor is it to be administered to humans or
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uct. The buyer agrees to use NuGEN products accompanying the product insert in accordance
with the intended use and the written instructions provided.
Table of Contents
Contents
I. Introduction .........................................................................................................1
A. Background .......................................................................................................1
B. How the Ovation One-Direct System Works .....................................................1
C. Performance Specifications ...............................................................................2
D. Quality Control .................................................................................................2
E. Storage and Stability .........................................................................................2
F. Material Safety Data Sheet (MSDS) ...................................................................3
II. Kit Components ..................................................................................................4
A. Reagents Provided ............................................................................................4
B. Additional Equipment, Reagents and Labware ................................................5
III. Planning the Experiment .....................................................................................7
A. Input RNA Requirements ..................................................................................7
B. Using RNase-free Techniques ...........................................................................9
C. RNA Storage .....................................................................................................9
D. Amplified cDNA Storage ..................................................................................9
IV. Protocol .............................................................................................................10
A. Overview .........................................................................................................10
B. Protocol Notes ................................................................................................10
C. Agencourt® RNAClean® XP Purification Beads ...............................................11
D. Programming the Thermal Cycler ...................................................................13
E. First Strand cDNA Synthesis ...........................................................................14
F. Second Strand cDNA Synthesis ......................................................................16
G. Purification of cDNA .......................................................................................17
H. SPIA Amplification ..........................................................................................18
I. Post-SPIA Modification ...................................................................................19
J. Purification of Amplified cDNA Protocol ........................................................20
K. Measuring cDNA Product Yield and Purity .....................................................22
V. Technical Support ..............................................................................................23
VI. Appendix ...........................................................................................................24
A. Considerations for Microarray Analysis ...........................................................24
B. Performing Quantitative PCR on Amplified cDNA .........................................24
C. Alternative Purification Protocols for Amplified cDNA ...................................24
D. Quality Control of Amplified cDNA Product ...................................................26
E. DNase Treatment of RNA................................................................................26
F. Preventing Non-specific Amplification ............................................................29
G. Frequently Asked Questions (FAQs) ...............................................................31
H. Update History ................................................................................................35
I. Introduction
A. Background
The Ovation® One-Direct System provides a fast and simple method for preparing
amplified cDNA for global gene expression analysis directly from a cell lysate prepared
from one or more cells, or purified total RNA samples as small as 10 pg. Amplification
is initiated at the 3’ end as well as randomly throughout the whole transcriptome in
the sample. This feature, along with highly refined amplification chemistry, makes the
Ovation One-Direct System ideal for amplification of the smallest biological samples
down to the single-cell level.
The amplified product of the Ovation One-Direct System is optimized for robust tran-
script detection using microarrays or real-time quantitative PCR (qPCR). The Ovation
One-Direct System can be used with Affymetrix GeneChip® arrays, Agilent Dual-Mode
Gene Expression microarrays or Illumina Whole-Genome Expression BeadChips
utilizing the appropriate NuGEN fragmentation and labeling modules and protocols.
Reagents for fragmentation and labeling are not included in the Ovation One-Direct
System and will need to be purchased separately. For details please visit the NuGEN
website at www.nugen.com.
The Ovation One-Direct System is powered by Ribo-SPIA® technology, a rapid, simple
and sensitive RNA amplification process developed by NuGEN. Using Ribo-SPIA tech-
nology and starting with a cell lysate from one or more cells, or 10 to 500 pg purified
total RNA, microgram quantities of cDNA can be prepared in approximately 8 hours.
The Ovation One-Direct System (Part No. 3500) provides optimized reagent mixes and
a protocol to process twelve RNA amplifications. Control RNA is not provided with the
Ovation One-Direct System but we strongly recommend the use of a control RNA when
using this product due to the inherent challenges in working with ultra-small biological
samples.
B. How the Ovation One-Direct System Works
The Ovation One-Direct System utilizes Ribo-SPIA technology that produces amplified
cDNA from total RNA (see Figure 1).
1. Generation of First Strand cDNA (1.5 hours)
First strand cDNA is prepared from a cell lysate or total RNA sample using a
unique first strand DNA/RNA chimeric primer mix and reverse transcriptase (RT).
The primers have a DNA portion that hybridizes either to the 5´ portion of the poly
(A) sequence or randomly across the transcript. RT extends the 3´ DNA end of each
primer generating first strand cDNA. The resulting cDNA/mRNA hybrid molecule
contains a unique RNA sequence at the 5´ end of the cDNA strand.
2. Generation of a DNA/RNA Heteroduplex Double Stranded cDNA (2 hours)
Fragmentation of the mRNA within the cDNA/mRNA complex creates priming
sites for DNA polymerase to synthesize a second strand, which includes DNA
complementary to the 5´ unique sequence from the first strand chimeric primers.
The result is a double stranded cDNA with a unique DNA/RNA heteroduplex at
one end.
1 Ovation One-Direct System
I. Introduction
3. SPIA® Amplification (2 hours)
SPIA amplification is an isothermal DNA amplification process developed by
NuGEN. The process uses a SPIA DNA/RNA chimeric primer, DNA polymerase and
RNase H in a homogeneous isothermal assay that provides highly efficient amplifi-
cation of DNA sequences. RNase H degrades RNA in the DNA/RNA heteroduplex
at the 5´ end of the first cDNA strand. This exposes a DNA sequence that is avail-
able for binding the SPIA DNA/RNA chimeric primer. DNA polymerase initiates
replication at the 3´ end of the primer, displacing the existing forward strand. The
RNA portion at the 5´ end of the newly synthesized strand is again removed by
RNase H, exposing the unique priming site for initiation of the next round of cDNA
synthesis. The process of SPIA DNA/RNA primer binding, DNA replication, strand
displacement and RNA cleavage is repeated, resulting in rapid accumulation of
cDNA with sequence complementary to the original mRNA.
4. Post-SPIA Modification (2 hours)
The Post-SPIA Modification process completes the One-Direct amplification
process. The first step allows the random primers to anneal to the single-stranded,
anti-sense cDNA target. The second step utilizes DNA polymerase to extend the
annealed primers and generate sense-target cDNA, producing targets appropriate
for either sense or anti-sense array.
C. Performance Specifications
The Ovation One-Direct System synthesizes microgram quantities of amplified cDNA
starting with cell lysate or total cellular RNA input amounts of 10 pg to 500 pg. In
approximately 7.5 hours, the system produces sufficient cDNA for fragmentation and
labeling, and subsequent hybridization to a microarray. Alternatively, the amplified
product may be used for qPCR reactions. When used with intact input RNA the size of
the majority of the cDNA products produced by the amplification process is between
50 bases and 500 bases.
D. Quality Control
Each Ovation One-Direct System lot is tested to meet specifications of yield, qPCR and
array performance.
E. Storage and Stability
Store First Strand and SPIA The Ovation One-Direct System is shipped on dry ice and should be unpacked imme-
Primer at –80°C. diately upon receipt. Note: This product contains components with multiple storage
temperatures.
The vials labeled First Strand Primer Mix (blue: A1) and SPIA Primer Mix (red: C1) should
be removed from the shipping carton upon delivery and stored separately at –80°C.
Store the RNAClean XP
Beads at 4°C The vial labeled Agencourt® RNAClean® XP Beads (clear cap) should be removed from
the top of the shipping carton upon delivery and stored at 4°C.
2 Ovation One-Direct System
I. Introduction
All remaining components should be stored at –20°C on the internal shelves of a freezer
without a defrost cycle.
After the first thaw, the Direct Lysis Buffer should be stored at 4°C. Do not refreeze the
Lysis Buffer.
The Ovation One-Direct System has been tested to perform to specifications for up to
six freeze/thaw cycles. Kits handled and stored according to the above guidelines will
perform to specifications for at least six months. We have not yet established long-term
storage conditions for the Ovation One-Direct System.
F. Material Safety Data Sheet (MSDS)
An MSDS for this product is available on the NuGEN website at
www.nugen.com/nugen/index.cfm/support/user-guides/.
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II. Kit Components
A. Reagents Provided
Table 1. First Strand cDNA Reagents
3500-12
COMPONENT VIAL CAP VIAL NUMBER
PART NUMBER
First Strand Primer Mix S01250 Blue A1 ver 5
First Strand Buffer Mix S01248 Blue A2 ver 5
First Strand Enzyme Mix S01249 Blue A2 ver 3
Table 2. Second Strand cDNA Reagents
3500-12
COMPONENT VIAL CAP VIAL NUMBER
PART NUMBER
Second Strand Buffer Mix S01176 Yellow B1 ver 3
Second Strand Enzyme Mix S01126 Yellow B2 ver 2
Table 3. SPIA Reagents
3500-12
COMPONENT VIAL CAP VIAL NUMBER
PART NUMBER
SPIA Primer Mix S01251 Red C1 ver 7
SPIA Buffer Mix S01164 Red C2 ver 5
SPIA Enzyme Mix S01165 Red C3 ver 5
4 Ovation One-Direct System
II. Kit Components
Table 4. Post-SPIA Modification Reagents
3500-12
COMPONENT VIAL CAP VIAL NUMBER
PART NUMBER
Primer Mix S01252 Red E1 ver 1
Buffer Mix S01253 Red E2 ver 2
Enzyme Mix S01254 Red E3 ver 1
Table 5. Additional Reagents
3500-12
COMPONENT VIAL CAP VIAL NUMBER
PART NUMBER
Nuclease-free Water S01001 Green D1
Direct Lysis Buffer S01255 Clear —
Agencourt RNAClean XP
S01307 Clear —
Beads
Note: The reagents in the Ovation One-Direct System product are similar to reagents
in NuGEN’s other kits. However, unless the part numbers are identical, these reagents
do not have exactly the same composition and therefore are not interchangeable. Do
not exchange reagents between different kits as it will adversely affect performance.
B. Additional Equipment, Reagents and Labware
Required Materials
• Equipment
- Microcentrifuge for individual 1.5 mL and 0.5 mL tubes
- Microcentrifuge or centrifuge for individual 0.2 mL tubes, strip tubes and
PCR  plates
- 0.5–10 µL pipette, 2–20 µL pipette, 20–200 µL pipette, 200–1000 µL pipette
- Vortexer
- Thermal cycler with 0.2 mL tube heat block, heated lid, and 100 µL
reaction capacity
- Appropriate spectrophotometer and cuvettes, or Nanodrop® UV-Vis
Spectrophotometer
5 Ovation One-Direct System
II. Kit Components
• Reagents
- Ethanol (Sigma-Aldrich, Cat. # E7023), for purification steps
• Supplies and Labware
- Nuclease-free pipette tips
- Low-retention microcentrifuge tubes (SafeSeal Low Binding 0.65 mL
Microcentrifuge Tubes, Sorenson Biosciences, Inc., Cat. #11300)
- 1.5 mL and 0.5 mL RNase-free microcentrifuge tubes
- 0.2 mL individual thin-wall PCR tubes or 8 X 0.2 mL strip PCR tubes or
0.2 mL thin-wall PCR plate
- Magnetic separation device
- QIAGEN MinElute® Spin Columns (Cat. #28204)
- Disposable gloves
- Kimwipes
- Ice bucket
• Optional materials
- Agilent 2100 bioanalyzer or materials and equipment for electrophoretic
analysis of RNA
- Real-time PCR system
- Decontamination solutions such as RNaseZap® (Ambion, Cat.# AM9780) and
DNA-OFF™ (MP Biomedicals, Cat.# QD0500)
To Order:
• Ambion Inc., www.ambion.com
• Beckman Coulter, www.beckmancoulter.com
• MP Biomedicals, www.mpbio.com
• New England BioLabs, www.neb.com
• QIAGEN Inc., www.qiagen.com
• Sigma-Aldrich, Inc., www.sigmaaldrich.com
• USB Corporation, www.usbweb.com
• Zymo Research, www.zymoresearch.com
• Sorenson Biosiences, www.sorbio.com
6 Ovation One-Direct System
III. Planning the Experiment
A. Input RNA Requirements
1. RNA Quantity
Ovation One-Direct is designed to use lysates from single cells, or cell pellets
as input without further purification using the Direct Lysis Buffer included in the
kit. Given that the amount of total RNA contained in a single cell varies widely
based on cell type and other factors, some experimentation may be necessary to
determine the optimum input cell numbers and working limits for a given sample
type.
Purified total RNA samples can also be used as input. We have successfully tested
inputs in the range from 10 pg to 500 pg.
2. RNA Purity
Purified total RNA samples must be free of contaminating proteins and other
cellular material, organic solvents (including phenol and ethanol) and salts used in
many RNA isolation methods. Use of a commercially available system for preparing
small amounts of RNA that does not require organic solvents is recommended. If a
method such as TRIzol® is used, we recommend employing an additional column
purification step after isolation in order to remove any residual organics. One
measure of RNA purity is the ratio of absorbance readings at 260 and 280 nm. The
A260:A280 ratio for RNA samples of acceptable purity should be in excess of 1.8.
RNA samples with lower ratios may result in poor amplification.
3. RNA Integrity
The Ovation One-Direct System is designed to use cell lysate samples prepared
with Direct Lysis Buffer as input. Purified total RNA samples of high molecular
weight with little or no evidence of degradation will also amplify very well with this
product.
RNA integrity can be determined using the Agilent 2100 Bioanalyzer, RNA 6000
Nano LabChip® or RNA 6000 Pico LabChip®. The RNA Integrity Number (RIN)
calculation, available in the Bioanalyzer 2100 Expert Software, provides an index
of RNA quality that can be helpful in triaging purified RNA samples of varying
integrity prior to amplification. While it is impossible to guarantee satisfactory
results with all degraded samples, the One-Direct System may work with many
samples that are moderately or even severely degraded.
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