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Agilent Low RNA Input Linear Amplification Kit PLUS Agilent Low RNA Input Linear Amplification Kit (20 reactions) Protocol Optimized protocol for use with Agilent oligonucleotide microarrays Version 4...
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Notices © Agilent Technologies, Inc. 2006 Technical Support No part of this manual may be reproduced in any form Technical product support may be obtained by contact- or by any means (including electr...
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Contents Introduction 5 Kit Contents 6 Agilent Low RNA Input Linear Amplification Kit (5184-3523) 6 Agilent Low RNA Input Linear Amplification Kit PLUS, One-Color (5188-5339) 7 Agilent Low RNA Input L...
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Agilent Low RNA Input Fluorescent Linear Amplification Kit Introduction Agilent's Low RNA Input Linear Amplification kits generate fluorescent complimen- tary RNA (c RNA) for use with Agilent's oligon...
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Kit Contents Agilent Low RNA Input Linear Amplification Kit (5184-3523) Component Volume (µL) T7 Promoter primer 5X First strand buffer 195 0.1 M DTT 10 m M d NTP mix MMLV RT RNase OUT 4X Transcriptio...
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Agilent Low RNA Input Linear Amplification Kit PLUS, One-Color (5188-5339) Component Volume (µL) T7 Promoter primer 5X First strand buffer 195 0.1 M DTT 10 m M d NTP mix MMLV RT RNase OUT 4X Transcrip...
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Agilent Low RNA Input Linear Amplification Kit PLUS, Two-Color (5188-5340) Component Volume (µL) T7 Promoter primer 5X First strand buffer 195 0.1 M DTT 10 m M d NTP mix MMLV RT RNase OUT 4X Transcrip...
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Required Equipment Ice and ice bucket Powderless gloves UV-Vis scanning spectrophotometer and 0.1-m L volume quartz microcuvettes (1-cm path length) or the Nano Drop ND-1000 Spectrophotometer. We reco...
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General Procedural Notes Follow Biosafety Level 1 (BL1) safety rules. Our c DNA master mix procedure specifies reagent volumes for 1 reaction and 6.5 reactions. We have added an extra half-reaction to...
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Agilent Low RNA Input Fluorescent Linear Amplification Kit Fluorescent c RNA Synthesis Procedure For use with Agilent oligo microarrays The fluorescent c RNA synthesis procedure was optimized to work ...
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Workflow Time Table for Preparation of Fluorescent c RNA Table 1 Preparation of fluorescent c RNA c DNA Synthesis (Total time: 155 minutes) Step Temperature Time (minutes) Denature primer and template...
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Schematic of amplified c RNA procedure Figure 1 Schematic of amplified c RNA procedure.
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Before Getting Started The following reagents are used for generating fluorescently labeled c DNA from c RNA (red caps): d NTPs, CTP, Random hexamers, RNase 1 “A”. If you are generat- ing labeled c RN...
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RNA Preparation and Qualification High-quality RNA starting material is essential for successful microarray results. It is essential that your total or poly A+ RNA be of high quality. We recommend the...
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c DNA Synthesis from Total RNA (Time required: ~3 hours) Add total or poly A+ RNA to a 1.5 m L microcentrifuge tube in a volume of 8.3 µL or less. Samples should be diluted such that at least 2 µL is ...
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Table 2 c DNA Master Mix Component Volume (µL)/reaction Volume (µL)/ 6.5 reactions 5X First strand buffer 4.0 0.1 M DTT 2.0 10 m M d NTP mix 1.0 6.5 MMLV-RT 1.0 6.5 RNase OUT 0.5 3.3 Total volume 8.5 ...
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Fluorescent c RNA Synthesis: in vitro Transcription and Incorporation of Cyanine 3- or Cyanine 5-CTP (Time required: ~2.5 hours) Cyanine 3 is bright pink and cyanine 5 is bright blue. Both are light s...
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Purification of Amplified RNA We support the use of Qiagen's RNeasy mini spin columns for purifying amplified c RNA samples. Unincorporated dye-labeled nucleotides in the hybridization solu- tion sign...
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Quantitating c RNA Products A typical c RNA amplification reaction, starting with 50 ng of total RNA, with Agilent's kit will yield 0.2– 2.0 µg of c RNA (2.0– 4.0 µg when starting with 500 ng of total...
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Plant RNA Quality Consistent with all RNA handling procedures, be careful when working with plant RNA to avoid introducing nucleases that will degrade your sample. Before labeling, be sure to check th...
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High quality Arabidopsis RNA Degraded Arabidopsis RNA
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RNA Quality Data from a high quality total RNA preparation Although a wide variety of ribosomal peak heights and ratios exist across a broad range of RNA sample types, most high quality RNA samples sh...
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Data from a high quality m RNA preparation High quality m RNA run on the Agilent 2100 Bioanalyzer typically has the shape of a broad peak, with transcripts falling in the range of 500–9000 bases long....
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Optional Thermocycler Protocol Thermocycler Programs Store the following programs into your thermocycler: Program 1: 65°C for 10 minutes, 4°C forever Program 2: 40°C for 2 hours, 65°C for 15 minutes, ...
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c DNA Synthesis from Total RNA (Time required: ~3 hours) Add 50 to 1000 ng of total RNA to a 0.2 m L PCR tube or the well of a 96-well PCR plate in a volume of 8.3 µL or less. The total concentration ...
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Fluorescent c RNA Synthesis: in vitro Transcription and Incorporation of Cyanine 3- or Cyanine 5-CTP (Time required: ~2.5 hours) Immediately before use, make one Master Mix for each cyanine dye by add...
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Agilent Technologies Agilent Gene Expression Microarrays Web site: www.agilent.com/chem/dna [email protected] Information, descriptions, and specifica- tions are subject to change without not...
