High Performance Labeling Kits for cdna and Oligo Microarrays

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1 Agilent in Life Sciences > Genomics > Proteomics > Drug Discovery > Development > QA/QC High Performance Labeling Kits for cdna and Oligo Microarrays

2 Get more from one experiment Agilent has perfected the art of two-color labeling and microarray analysis. By using a two-color labeling protocol, you can be confident that you have: reduced the system variability between two samples and normalized the noise between two microarrays being studied. Reliability is further enhanced when researchers take advantage of running samples on Agilent s complete microarray-based gene expression system. Two-Color Labeling Agilent further enhances two-color labeling by including correction and dye bias algorithms specific to each type of Agilent microarray and its corresponding labeling method in its Feature Extraction Software which automatically extracts data and corrects background for each Agilent microarray being studied. Downstream gene expression data analysis using two-color data is facilitated with Rosetta Resolver or Luminator which also allows for incorporation of onecolor data. Reducing microarray experimental variability while enhancing overall performance and quality are hallmarks of Agilent s gene expression solution. High-quality differential expression experiments all in a single microarray Simple two-color (red vs. green) analysis Advanced gene expression studies using Rosetta Resolver or Luminator for 2-color differential expression analysis

3 On track to results with less sample Less Sample If you have abundant sample, then you ll want to use the simplest, fastest labeling procedure available Agilent s convenient Fluorescent Direct Label Kit. In experiments involving laser microdissected specimens, biopsy samples or challenging cell cultures, researchers need a complete labeling kit they can trust to amplify small amounts of starting material. Now there s an answer. Turn to Agilent s new Low RNA Input Fluorescent Linear Amplification Kit that requires as little as 50 ng of total RNA and only a single round of amplification...all in one tube. Consider Agilent s labeling kits. They re the answer you ve been looking for Discover what you ve been missing Good RNA preparation & QC are essential to providing the right starting material for any labeling experiment. But even if you have good starting material, you may not end up with labeled samples you can trust. That s why you need flexible, pre-configured kits that work with specific gene expression microarrays. Agilent s labeling kits provide added confidence with 2-color labeling formats that can normalize system variability between two samples and the noise between two microarrays. Couple this with Agilent s efficient labeling protocols and you have a labeling system that will put you on the right track to successful gene expression experimentation. Flexible Product Choices cdna labeling Agilent Fluorescent Direct Label Kit Agilent Low RNA Input Fluorescent Linear Amplification Kit crna labeling Agilent Fluorescent Linear Amplification Kit Agilent Low RNA Input Fluorescent Linear Amplification Kit

4 Convenience coupled with consistency Agilent labeling kits feature out-of-the-box ease of use. Each kit contains the necessary reagents to carry out cdna synthesis or crna synthesis saving you from having to purchase components separately from multiple vendors. You ll always be ready to label your samples with added confidence when you use Agilent s labeling kits. Cyanine dyes and routine lab reagents are all that you need to purchase separately. Labeling methods Prepare microarray targets for gene expression analysis by one of three robust Agilent labeling methods Method 1: cdna labeling Method 2: Amplified cdna labeling Method 3: Amplified crna labeling Agilent labeling kit Agilent Fluorescent Direct Label Kit (G2557A) Agilent Low Input RNA Fluorescent Linear Amplification Kit ( ) Agilent Low Input RNA Fluorescent Linear Amplification Kit ( ) Starting amount of RNA 10 µg total RNA or 200 ng poly A+ RNA* 50 ng total RNA 50 ng total RNA Labeling procedure Direct Labeling of cdna from poly A+ RNA via MMLV Reverse Transcriptase Double-stranded cdna is synthesized from poly A+ RNA via MMLV Reverse Transcriptase. Amplified crna is created via T7 RNA Polymerase, which is then the template for amplified cdna Double-stranded cdna is synthesized from poly A+ RNA via Reverse Transcriptase. Amplified labeled crna is created via T7 RNA Polymerase Purification Purify with Qiagen QIAquick spin columns Purify with Qiagen QIAquick spin columns Purify with Qiagen RNeasy mini spin columns Relative amount of labeled target 1X 100X 100X Hybridize to this type of Agilent microarray cdna or Oligo cdna Oligo * For Agilent Oligo and cdna Microarrays (2 microarrays/slide format), use 10 µg total RNA or 200 ng poly A+ RNA for each labeling reaction/microarray. For Agilent Oligo Microarrays (1 microarray/slide format), use 20 µg total RNA or 400 ng poly A+ RNA for each labeling reaction/microarray.

5 High performance from start to finish Agilent provides labeling kits for use in situations where you have either large or small amounts of sample to work with. For abundant samples (10 µg to 20 µg total RNA), use Agilent s Fluorescent Direct Label Kit for rapid and reliable labeling. And where sample is very limited (50 ng total RNA), turn to the recently developed Agilent Low RNA Input Fluorescent Linear Amplification Kit. It features a single round of amplification plus the flexibility of labeling both cdna and crna all in a single kit. All Agilent labeling kits have been tested with Agilent microarrays and include comprehensive protocols. Realize the benefits of fewer steps plus lower RNA input with Agilent labeling kits Products Fluorescent Direct Label Kit Fluorescent Linear Amplification Kit Low RNA Input Fluorescent Linear Amplification Kit Total RNA input 10 µg* 5 µg 50 ng Working range 5-25 µg µg ng Output target cdna crna cdna or crna Target yield ng 5-50 µg µg crna/50 ng input Reaction time 3-4 hours 8 hours cdna (~10 hours) crna (~6 hours) * For Agilent Oligo and cdna Microarrays (2 microarrays/slide format), use 10 µg total RNA or 200 ng poly A+ RNA for each labeling reaction/microarray. For Agilent Oligo Microarrays (1 microarray/slide format), use 20 µg total RNA or 400 ng poly A+ RNA for each labeling reaction/microarray.

6 cdna Labeling Agilent Fluorescent Direct Label Kit For use with all cdna and Oligo Microarrays The Agilent Fluorescent Direct Label Kit generates cyanine 3- or cyanine 5-labeled cdna targets from as little as 10 µg total RNA or 200 ng poly A+ RNA using minimal dye. This procedure can be completed quickly and involves only 3 steps: synthesizing fluorescent-labeled cdna using reverse transcriptase digesting the RNA cdna target purification Number of reactions included in kit Approximate RNA use/ microarray relative to Agilent microarray formats Overall time Highlights 20 reactions One reaction using 10 µg total RNA (2 microarrays per slide format); 20 µg total RNA for 1 microarray per slide format 3-4 hours (depends on number of samples) Kit benefits Simple protocol that takes only 3-4 hours depending on the number of samples Validated for use with both Agilent cdna and Oligo Microarrays for gene expression analysis. The kit may also be used with other types of microarrays. Sensitivity of 3 mrna copies per 10 6 cells using the complete Agilent Gene Expression System Minimal cyanine dye use while maintaining sensitivity Incorporation of cyanine 3- and cyanine 5-dCTP with similar efficiencies Single column purification for cyanine 3- and cyanine 5- labeled targets Protocol for using total or poly A+ sample RNA is included RNA sample requirement per labeling reaction Input RNA working range Labeling technique 10 µg total RNA, 200 ng poly A+ RNA* 5-25 µg total RNA Incorporation of dye-dctp by reverse transcriptase * For Agilent Oligo and cdna Microarrays (2 microarrays/slide format), use 10 µg total RNA or 200 ng poly A+ RNA for each labeling reaction/ microarray. For Agilent Oligo Microarrays (1 microarray/slide format), use 20 µg total RNA or 400 ng poly A+ RNA for each labeling reaction/ microarray.

7 Agilent Fluorescent Direct Label Kit Assay principles Total RNA containing poly A+ RNA or oligo (dt) purified poly A+ RNA is the template in the direct label protocol. A fluorescently-labeled cdna target is synthesized by adding Oligo dt Primer, MMLV-RT, dntps and cyanine labeled dctp s. After cdna synthesis, the RNA template is degraded by adding RNase I A. The cyanine 3- and cyanine 5- labeled samples are then combined and hybridized to a microarray. Microarray features common to both samples will appear yellow after hybridization. Those features common to Sample A will appear red (cyanine 5), and those common to Sample B (cyanine 3) will appear green. Number of features in bin Fluorescent signal distribution in a HeLa self vs. self hybridization (input RNA: total RNA) Cyanine 3 and cyanine 5 are incorporated with similar efficiencies Data generated using the Agilent Fluorescent Direct Label Kit to synthesize fluorescentlylabeled targets; targets were then hybridized to the Agilent Human 1 cdna microarray (G4100A). Each bin represents the fluorescent intensity of the raw signal less background within the indicated range. No normalization of the fluorescent signal has been performed. Fluorescent signal bin

8 Log ratio correlation between 20 µg and lower amounts of total RNA input Correlation coefficient (R 2 ) Total RNA input (µg) Validated performance Samples comprising varying amounts of total RNA were tested to ascertain the lowest possible amount of starting material that could be reliably used in a labeling reaction using Agilent s Fluorescent Direct Label Kit and the Agilent Human 1 cdna Microarray Kit (G4100A). The log ratio correlation plot demostrates that total RNA starting material representing 5 µg and above can be reliably used in this labeling reaction. All Agilent labeling kits go through the same rigorous pre-testing to determine guidance specifications for end-users. Agilent Fluorescent Direct Label Kit Kit contents MMLV-RT 5X First Strand Reaction Buffer 0.1 M DTT DNA primer dntp mix (-dctp) 5 mm dctp RNase I A Protocol for poly A+ RNA and total RNA Compatibility with Agilent Microarrays The Agilent Direct Label Kit is compatible with and has been validated for use with all Agilent cdna and Oligonucleotide Microarrays. Ordering information Agilent Fluorescent Direct Label Kit (G2557A) Non-Agilent components needed Vendor Part number Cyanine 3-dCTP PE-NEN NEL 576 Cyanine 5-dCTP PE-NEN NEL 577 QIAquick PCR Purification Kit (50) Qiagen For a discount on dyes, refer to quote AG2001 when ordering from PerkinElmer/NEN

9 crna Labeling cdna Labeling Highlights Agilent Low RNA Input Fluorescent Linear Amplification Kit This next generation linear amplification procedure delivers the benefits of single tube reactions found in the Fluorescent Linear Amplification kit...but now offers low RNA sample input combined with the ability to label both cdna and crna all in one kit.* The Agilent Low RNA Input Fluorescent Linear Amplification Kit makes a cyanine 3- or cyanine 5-labeled crna for oligo microarrays or a cyanine 3- or cyanine 5-labeled cdna for cdna microarrays from as little as 50 ng total RNA. The simple, patented, single-tube procedure consists of converting mrna primed with an oligo (d)t-t7 primer into dsdna with MMLV-RT, and then generates amplified crna using T7 RNA Polymerase. The kit also contains the needed reagents to convert crna to fluorescent cdna. The amplification reaction has been optimized to be linear and not introduce bias of the abundant mrna species over the rare mrna populations. The hybridized target/ probe signals on the microarray can then be visualized using Agilent s dual-color scanner or a comparable imaging system. Number of reactions included in kit Overall time RNA sample requirement per labeling reaction Type of labeling Labeling technique Consistent crna yield Sensitivity 20 reactions Oligo microarrays crna labeling (6 hours) cdna microarrays cdna labeling (10 hours) (depends on number of samples) 50 ng total RNA Cyanine 3 & cyanine 5 fluorescent labeling Linear amplification using MMLV-RT and T7 polymerase 100x minimum, typical x amplification depending on the sample As little as 1 mrna copy per 10 4 cells using the complete Agilent gene expression solution Kit benefits Simple protocol that provides you with the flexibility of generating either labeled cdna or crna Minimal hands-on time facilitated by a simple SINGLE TUBE procedure that is amenable to automation Comparable amplification to original Eberwine procedure 100x minimum, x typical amplification, but with fewer purification steps (no phenol extraction) and fewer reagents Validated for use with Agilent cdna or Oligo Microarrays; can also be used with other microarrays Produces approximately µg crna/50 ng input depending on sample type *Cyanine dyes and an appropriate Qiagen prep kit need to be ordered separately.

10 Amplified crna and cdna Labeling 50 ng 1 tube Less sample, fewer steps...

11 ...all in one kit

12 Agilent Low RNA Input Fluorescent Linear Amplification Kit Average log ratio across 4 microarrays (+/- 1SD) Spike-in s at different amounts Original mrna ratios maintained after one round of amplification An mrna transcript was spiked into the amplification reaction at various amounts from 20 to 2500 fg (at a ratio of 1 to 5) into 50 ng of HeLa Total RNA labeled with cyanine 3 and 50 ng of Spleen Total RNA labeled with cyanine 5. The log ratio of red (cyanine 5) to green (cyanine 3) was expected to be 0.7. The actual log ratio measurements of the spiked-in transcripts (4 replicates per sample) across the concentration range is close to the expected value demonstrating that the ratio is maintained after one round of amplification. Low RNA input capability enables cancer research Agilent s Low RNA Input Fluorescent Linear Amplification kit was used to study gene expression of sample limited melanoma samples compared to normal specimens.* Approximately ,000 CD8+ T cells were isolated from healthy patient PBMCs (peripheral blood mononuclear cells). Total RNA was extracted and labeled with the kit. Fluorescently-labeled targets were hybridized to Agilent Human 1 cdna Microarrays. The data below demonstrate efficient 2-color labeling and low levels of noise in the system. Log 10 (intensity cyanine 5) Log 10 (intensity cyanine 3) Figure 1. Microarray data from a self-self experiment hybridized with cyanine 3- and cyanine 5-labeled cdna amplified from ,000 CD8+ T cells. Data points represent the red signal versus the green signal for each microarray. Data converges to a tight line corresponding to the expected log ratio of 0, indicating that the signal from the cyanine 3 labeling reaction was identical to that of the cyanine 5 reaction. CD8+ RNA vs. Reference Log 10 (query ratio) Figure 2. Depicts combined microarray data from two microarrays representing the two halves of a dye swap experiment. In one experiment, the microarray was hybridized with cyanine 3-labeled CD8+ T cell cdna and cyanine 5-labeled Human Reference Sample cdna. In the second experiment, the microarray was hybridized with cyanine 5-labeled CD8+ T cell cdna and cyanine 3-labeled Human Reference Sample cdna. Together, the experiments represent the two polarities in a dye swap experiment. Less than 1 percent of the genes were found to be anti-correlated while 97 percent were correlated, demonstrating the reproducibility of the labeling procedure. *Part of a collaborative study between Stanford University and Agilent Technologies.

13 crna Yield (µg) in 8 replicates (+/-1 SD) Amplification of Spleen and HeLa crnas from three users Amplification performance with just 50 ng of total RNA Data was generated from three researchers using Agilent s Low RNA Input Fluorescent Linear Amplification Kit. Users 1 and 2 ran the kit for the first time. User 3 had extensive experience using this kit. With very little starting material (50 ng Total RNA) from Spleen and HeLa specimens, all researchers were able to achieve consistent and reproducible yields with this efficient single tube amplification procedure. Agilent Low RNA Input Fluorescent Linear Amplification Kit Kit contents T7 Promoter Primer 5X First Strand Reaction Buffer 0.1 M DTT 10 mm dntp mix MMLV-RT (quantity 2) RNaseOUT 4X Transcription Buffer NTP Mix Inorganic Pyrophosphatase T7 RNA Polymerase Random Hexamers dntp Mix RNase I A CTP PEG All Components are RNase-free, DNase-free and of molecular biology grade or higher. Compatibility with Agilent Microarrays The Agilent Low RNA Input Fluorescent Linear Amplification Kit is compatible with all Agilent Oligonucleotide Microarrays and can also be used with Agilent s cdna Microarrays. Ordering information Agilent Low RNA Input Fluorescent Linear Amplification Kit* ( ) *US Patent Number US A1, Shannon, Karen W., issued 17 October 2001 Non-Agilent components needed Vendor Part number Cyanine 3-dCTP(cDNA labeling) PE-NEN NEL 576 Cyanine 5-dCTP (cdna labeling) PE-NEN NEL 577 Cyanine 3-CTP (crna labeling) PE-NEN NEL 580 Cyanine 5-CTP (crna labeling) PE-NEN NEL 581 RNeasy (crna labeling) Qiagen QIAquick (cdna labeling) Qiagen For a discount on dyes, refer to quote AG2001 when ordering from PerkinElmer/NEN

14 crna Labeling Agilent Fluorescent Linear Amplification Kit Agilent s first generation linear amplification kit.* Loaded with industry-firsts that include single tube amplification for reduced variability in labeling. Number of reactions included in kit Highlights 20 reactions The Agilent Low RNA Input Fluorescent Linear Amplification Kit makes a cyanine 3- or cyanine 5-labeled crna target from mrna from as little as 5 µg total RNA. The simple, patented, single-tube procedure consists of converting mrna primed with an oligo (d)t-t7 primer into dsdna with MMLV-RT, and then amplifying using T7 RNA Polymerase. Agilent s 2-color labeling procedure reduces system variability and noise between two microarrays. Another benefit is highly efficient amplification in just one cycle, thereby making the reaction very linear and representative of the sample being amplified. The cyanine 3- and cyanine 5-labeled crnas are used as targets on Agilent s Oligo Microarrays which can be scanned on Agilent s dual-color scanner or other compatible imaging system. Overall time RNA sample requirement per labeling reaction Type of labeling Labeling technique Consistent cdna yield Sensitivity Oligo microarrays crna labeling (5 hours polya+ RNA) or (8 hours total RNA) depends on number of samples 5 µg total RNA, 200 ng poly A+ RNA Cyanine 3 & cyanine 5 fluorescent labeling Linear amplification using MMLV-RT and T7 polymerase 100x minimum, typical x amplification, depending on sample As little as 1 mrna copy per 10 6 cells using the complete Agilent gene expression solution Kit benefits Simple protocol (as shown on page 11) with minimal steps...reduces hands-on time Single tube procedure amenable to automation Comparable amplification to original Eberwine procedure 100x minimum, typical x amplification No phenol extraction step Optimized protocol for use with poly A+ RNA or total RNA Validated for use with Agilent Oligo Microarrays; can also be used with other microarrays *Cyanine dyes and an appropriate Qiagen prep kit need to be ordered separately

15 Agilent Fluorescent Linear Amplification Kit Demonstrates similar mrna ratios after amplification A major issue with amplifying mrna is maintaining the same representation of the original mrna pool. To test this using the Agilent Linear Amplification Kit, three different plant mrnas were spiked into a pool of mammalian mrnas, amplified, and concentrations measured using radiolabeled hybridization with plant mrna probes. This was then compared with the total RNA amplification levels. The results (Figure 1) reveal that the spiked transcripts in the mrna pool were amplified to the same extent as the total mrna pool. In addition, transcripts spiked in at different concentrations were amplified to the same extent as the total mrna population, showing that the amplification is linear. Amplification in this experiment was approximately 250-fold. Antisense RNA produced (fmols) Figure Sense RNA input (fmols) Exceptional linearity across a wide dynamic range Polyadenylated RNAs were produced in vitro from cloned bacterial genes. These transcripts were then spiked into 200 ng HeLa cell poly(a)+ RNA at different concentrations and the mixture was then amplified using the Agilent Fluorescent Linear Amplification Kit. For each of the resulting amplification reactions, 0.3 µg were hybridized to Agilent 60-mer Oligonucleotide Microarrays containing probes for the bacterial sequences. The plot (Figure 2) shows the amount of labeled spike-in transcript per amplification reaction (labeled with cyanine 5- CTP) vs. the red (cyanine 5) background-subtracted hybridization signal. The signal shows the linearity of the amplification reaction with respect to the amount of input spike-in. rbgsubsignal Figure 2 Bacterial spike-in #1 vs. rbgsubsignal pg input cyanine 5-labeled spike-in

16 Agilent Fluorescent Linear Amplification Kit Kit contents T7 Promoter Primer 5X First Strand Reaction Buffer 0.1 M DTT 10 mm dntp mix Random Hexamers MMLV-RT RNaseOUT 4X Transcription Buffer NTP Mix Inorganic Pyrophosphatase T7 RNA Polymerase 4 M LiCl All Components are RNase-free, DNase-free and of molecular biology grade or higher. Compatibility with Agilent Microarrays The Agilent Fluorescent Linear Amplification Kit is compatible with all Agilent Oligo Microarrays. The kit is NOT compatible with Agilent cdna Microarrays. Ordering information Agilent Fluorescent Linear Amplification Kit* (G2554A) *US Patent Number US A1, Shannon, Karen W., issued 17 October 2001 Non-Agilent components needed Vendor Part number Cyanine 3-CTP PE-NEN NEL 580 Cyanine 5-CTP PE-NEN NEL 581 For a discount on dyes, refer to quote AG2001 when ordering from PerkinElmer/NEN

17 Product information and online shopping just a click away Agilent s comprehensive website offers technology buyers the most up-to-date information on new products as well as existing products and services. Register online to receive monthly updates with our new e-notes. Agilent makes purchasing its microarrays, labeling and hybridization reagents and accessories easy with its online store at:

18 Sample preparation. Do more with less. Get more out of your sample preparation steps. QC your samples and label them efficiently with Agilent s full line of sample preparation tools. They deliver performance and productivity enhancements that also translate to better quality results. That s because they have been exhaustively tested and validated to work seamlessly with one another on Agilent s gene expression product line-up. Another good reason to consider doing more with less... and all with Agilent. Step One: QC it to be sure about it. Agilent s 2100 bioanalyzer and related RNA 6000 Pico and Nano LabChip kits are rapidly becoming the defacto standard for RNA QC prior to running microarray experiments. Researchers worldwide have witnessed the ease-of-use and high quality, objective results for themselves. To learn more about these and other Agilent lab-on-a-chip products, visit: Step Three: Evaluate it with ease. Agilent developed the world s first benchtop diode-array UV-vis spectrophotomether, the 8400 series. Researchers trust its performance to deliver accurate UV analysis of RNA samples. However, if you have very little sample to analyze, Agilent s labeling protocols have been optimized to include other options for post-labeling QC analysis of samples. Agilent is committed to making sure your microarray and other gene expression experiments will deliver results, time and time again. Step Two: Label it with confidence. In this brochure, you ll be enlightened about new ways to achieve flexibility and reliability in labeling procedures. Learn about Agilent s Direct and Linear Amplification procedures and reagent kits that have been designed to meet the needs of researchers who demand out-ofthe-box ease-of-use, validated labeling procedures that match specific microarray formats as well as sample sparing protocols that enable experimentation on very little sample.

19 Agilent Gene Expression Solutions an integrated, flexible and modular approach to microarray-based research Agilent s flexible gene expression solutions give you the freedom to drive your research where you want it to go, getting you miles ahead of the competition. Discover what you ve been missing with Agilent s open platform approach. Grows to meet your needs It s modular and integrated based on a standard microarray 1 x 3 glass slide format Buy just what you need today Add products and services later, knowing that they all work together Find out more at Agilent.com Agilent continues to add value-add products and services to its comprehensive, integrated gene expression solutions. For more detailed, up-to-date information on Agilent s full line of Gene Expression solutions, visit Or to buy online, simply log on to

20 Ordering information u.s. and canada japan asia pacific: adinquiry_aplsca@agilent.com europe: marcom_center@agilent.com global: dna_microarray@agilent.com Agilent Technologies, Inc Research Use Only Rosetta Resolver is a U.S. registered trademark of Rosetta Inpharmatics. Luminator is a trademark of Rosetta Inpharmatics. Information, descriptions and specifications in this publication are subject to change without notice. Printed in the U.S.A. April 15, EN