ABI PRISM 7000 Sequence Detection Systems Relative ...

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Primer Extended on mRNA 5′ 3′ 5′ 3′ 5′ Synthesis of 1st cDNA strand Reverse Primer cDNA Oligo d(T) or random hexamer cDNA Have you: • Selected the PCR method • Designated the four essential components • Selected an SDS chemistry & reagent configuration • Designed primers and probes Yes No Chapter 2 Design an RQ experiment according to guidelines in chapter 2. Do you have purified total RNA A C U U GU No page 16 Isolate total RNA using these guidelines. Yes Is the concentration of RNA within the recommended range .1 to 10 µg No page 16 Adjust RNA concentration. Yes Have you converted the total RNA to cDNA on mRNA DNA strand 5′ cDNA Reverse Primer Oligo d(T) or random hexamer 5′ cDNA 3 No page 16 Go to chapter 4, Generating Data from RQ Plates Convert total RNA to cDNA using the High Capacity cDNA Archive Kit. DRAFT September 26, 2003 3:14 pm, C3_RT.fm Chapter 3 Performing Reverse Transcription
Primer Extended on mRNA 5′ 3′ 5′ cDNA Reverse Primer Oligo d(T) or random hexamer Synthesis of 1st cDNA strand 3′ 5′ cDNA Performing Reverse Transcription Primer Extended on mRNA 5′ 3′ 5′ cDNA Reverse Primer Oligo d(T) or random hexamer Synthesis of 1st cDNA strand 3′ 5′ cDNA Workflow Designing an RQ Experiment Performing Reverse Transcription Generating Data from RQ Plates Performing Reverse Transcription (cDNA generation) 1. Prepare total RNA samples. 2. Use the manual method of the High Capacity cDNA Archive Kit to generate cDNA. 3 Performing an RQ Study RT-PCR Methods DRAFT September 26, 2003 3:14 pm, C3_RT.fm Notes As discussed in “Selecting the PCR Method” on page 7, there are two RT-PCR methods that you can choose from when performing RQ experiments: one-step RT-PCR or twostep RT-PCR. IMPORTANT! Applied Biosystems recommends that you use the two-step RT-PCR method for RQ experiments. This guide assumes that RQ experiments are designed using two-step RT-PCR. Further, only the recommended reagent configurations are documented. For additional options, refer to the Sequence Detection Systems Chemistry Guide. Relative Quantification Getting Started Guide for 7000 v1.1 15
Page 1 and 2: 5′ cDNA Reverse Primer Oligo d(T)
Page 3 and 4: Primer Extended on mRNA 5′ 3′ 5
Page 5 and 6: Chapter 5 Performing an RQ Study 33
Page 7 and 8: Preface How to Use This Guide Purpo
Page 9 and 10: Safety and EMC Compliance Informati
Page 11 and 12: Symbols on Instruments Electrical S
Page 13 and 14: Chemical Safety Chemical Hazard War
Page 15 and 16: Physical Hazard Safety Ultraviolet
Page 17 and 18: Safety and Electromagnetic Compatib
Page 19 and 20: Introduction 1 About the 7000 SDS I
Page 21 and 22: About the Sample RQ Experiment Desc
Page 23 and 24: Materials and Equipment Description
Page 27 and 28: Specifying the Components of an RQ
Page 29 and 30: Selecting the Sequence Detection Ch
Page 31: Choosing the Probes and Primers Abo
Page 35 and 36: 5′ 3′ 5′ cDNA Reverse Primer
Page 39 and 40: Preparing the PCR Master Mix Preven
Page 41 and 42: Creating an RQ Plate Document Detec
Page 43 and 44: Specifying Thermal Cycling Conditio
Page 45 and 46: Analyzing and Viewing RQ Plate Data
Page 47 and 48: Analyzing and Viewing RQ Plate Data
Page 49 and 50: Exporting RQ Plate Documents Reanal
Page 53 and 54: Creating an RQ Study Document 5. Co
Page 55 and 56: Analyzing and Viewing the Results o
Page 57 and 58: Analyzing and Viewing the Results o
Page 59 and 60: Omitting Samples from a Study Omitt
Page 61 and 62: Exporting RQ Study Results Exportin
Page 63 and 64: References Kwok, S. and Higuchi, R.
Page 65 and 66: Creating Detectors A Before you can
Page 67 and 68: Upgrading the 7000 Software with RQ
Page 69 and 70: Index SAMPLE DOCUMENT September 26,
Page 71 and 72: Index SAMPLE DOCUMENT September 26,
Page 73 and 74: DRAFT September 26, 2003 3:17 pm, G

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