GeneChip Expression 3 - Amplification Reagents

Transcription

1 GeneChip Expression 3 - Amplification Reagents Two-Cycle Target Labeling Assay and Two-Cycle cdna Synthesis Kit July 2004

2 Outline Overview Procedural modifications Technical performance compared with a previous small sample target labeling protocol (SSTLvII( SSTLvII) Technical performance compared with the standard One-Cycle Target Labeling Assay Summary

3 GeneChip Target Labeling Assays Total RNA Starting Materials mrna Target Labeling Assay 1 15 µg µg One-Cycle ng N/A Two-Cycle

4 Two-Cycle cdna Synthesis Kit

5 A Complete Reagent Solution for GeneChip Expression Analysis

6 Two-Cycle Target Labeling and Control Reagents Two-Cycle Target Labeling and Control Reagents (30 reactions) P/N Two-Cycle cdna Synthesis Kit IVT Labeling Kit Sample Cleanup Module Poly-A A RNA Control Kit Hybridization Controls Note: MEGAscript T7 Kit needs to be ordered directly from Ambion for the intermediate IVT step with unlabeled nucleotides.

7 A Complete GeneChip Reagent Solution for Expression Analysis Includes both target labeling and control reagents The High-Resolution GeneChip Scanner 3000, new Fluidics Station 450, 11-µm m feature size arrays, and these new reagents represent the next-generation system offering the best performance for expression analysis Provide consistency, convenience, ease of use, and standardization for GeneChip experiments

8 Two-Cycle cdna Synthesis Kit Component Name Concentration Volume Quantity T7-Oligo(dT) Primer, 50 µm 50 µm 120 µl 1 5X 1 st Strand Reaction Mix 5X 180 µl 1 DTT, 0.1M 0.1M 90 µl 1 dntp, 10 mm 10 mm 200 µl 1 SuperScript TM II 200 U/µL 60 µl 1 RNase Inhibitor 40 U/µL 45 µl 1 5X 2 nd Strand Reaction Mix 5X 900 µl 1 Random Primers 3 µg/µl 60 µl 1 MgCl 2, 1M 1M 60 µl 1 E.coli DNA Polymerase I 10 U/µL 200 µl 1 RNase H 2 U/µL 55 µl 1 T4 DNA Polymerase 5 U/µL 60 µl 1 RNase-free Water 9.5 ml 1 30 reactions/kit packaging Shelf life = 18 months

9 Two-Cycle cdna Synthesis Kit Features Manufactured by Invitrogen for Affymetrix, uniquely configured and validated for GeneChip assay Provides essential reagents in one convenient kit for two cycles of cdna synthesis Designed for applications with limited starting materials of ng of total RNA Based on a proven, robust, and streamlined protocol completing target labeling in only 2 ½ days for increased success of array experiments Customers do need to purchase MEGAscript reagents kit directly from Ambion for the IVT step in the first cycle

10 Outline Overview Procedural modifications Technical performance compared with a previous small sample target labeling protocol (SSTLvII( SSTLvII) Technical performance compared with the standard One-Cycle Target Labeling Assay Summary

11 Streamlined Protocol for Improved Success Reducing experimental time to 2 ½ days No cdna cleanup step in the 1 st cycle No precipitation or speed vacuum Increased reaction volume for 1 st cycle cdna synthesis for improved ease of use IVT reactions performed overnight

12 Heated Lid For 2 nd -Strand cdna Synthesis Reduced crna Yield with 10 ng of Total RNA crna Yield (µg) No Lid Avg Std. Dev With Lid Avg Std. Dev. 9.69

13 Outline Overview Procedural modifications Technical performance compared with a previous small sample target labeling protocol (SSTLvII( SSTLvII) Technical performance compared with the standard One-Cycle Target Labeling Assay Summary

14 Sufficient crna Yield From ng Total RNA 140 crna Yield (micrograms) SSTL (10 ng) 2-Cycle (10 ng) SSTL (100 ng) 2-Cycle (100 ng) 0 HeLa Human Brain Hybridized to 18-µm feature size arrays.

15 Global Detection Sensitivity Maintained 60% 50% % Present 40% 30% 20% 10% SSTL (10 ng) 2-Cycle (10 ng) SSTL (100 ng) 2-Cycle (100 ng) 0% HeLa Human Brain Hybridized to 18-µm feature size arrays.

16 3 /5 Ratios of Control Genes GAPDH Ratios 6 3'/5' Ratio HeLa Human Brain SSTL (10 ng) 2-Cycle (10 ng) SSTL (100 ng) 2-Cycle (100 ng) β-actin Ratios 3'/5' Ratio SSTL (10 ng) 2-Cycle (10 ng) SSTL (100 ng) Hybridized to 18-µm feature size arrays. 0 HeLa Human Brain 2-Cycle (100 ng)

17 Intra-Assay Reproducibility - High Signal Correlation 1.00 Signal Correlation Value SSTL (10 ng) 2-Cycle (10 ng) SSTL (100 ng) 2-Cycle (100 ng) 0.90 HeLa Human Brain Hybridized to 18-µm feature size arrays.

18 Intra-Assay Reproducibility - High Detection Call Concordance 100% Detection Concordance Value 95% 90% 85% 80% HeLa Human Brain SSTL (10 ng) 2-Cycle (10 ng) SSTL (100 ng) 2-Cycle (100 ng) Hybridized to 18-µm feature size arrays.

19 Intra-Assay Reproducibility - False Change 5.0% False Change Value 4.5% 4.0% 3.5% 3.0% 2.5% 2.0% 1.5% 1.0% 0.5% 0.0% HeLa Human Brain SSTL (10 ng) 2-Cycle (10 ng) SSTL (100 ng) 2-Cycle (100 ng) Hybridized to 18-µm feature size arrays

20 Outline Overview Procedural modifications Technical performance compared with a previous small sample target labeling protocol (SSTLvII( SSTLvII) Technical performance compared with the standard One-Cycle Target Labeling Assay Summary

21 Signal Correlation Compared With One-Cycle Target Labeling Assay 1.00 Signal Correlation Value HeLa Human Brain 1 µg. vs. 50 ng. 5 µg. vs. 50 ng.

22 High Call Concordance Between One- and Two-Cycle Target Labeling Assays HeLa Two-Cycle (50 ng) One-Cycle (1 µg) P A Human Brain Two-Cycle (50 ng) One-Cycle (1 µg) P A P 39.7% 7.6% P 46.2% 6.9% A 4.4% 48.3% A 4.9% 41.9% HeLa Two-Cycle (50 ng) One-Cycle (5 µg) P A Human Brain Two-Cycle (50 ng) One-Cycle (5 µg) P A P 40.7% 8.5% P 46.6% 7.2% A 3.4% 47.4% A 4.6% 41.7%

23 Outline Overview Procedural modifications Technical performance compared with a previous small sample target labeling protocol (SSTLvII( SSTLvII) Technical performance compared with the standard One-Cycle Target Labeling Assay Summary

24 Summary Two-Cycle cdna Synthesis Kit provides a convenient option for essential reagents used for Two-Cycle Target Labeling Consistent and robust crna yields for ng of total RNA Global detection sensitivity and reproducibility are maintained with the limited starting material Similar results are expected comparing the One- and Two- Cycle Target Labeling Assays Differences do exist between assays. For the most direct and simple interpretation, the same amount of starting material should be used in one project following a single assay protocol