ABI PRISM 7000 Sequence Detection Systems Relative ...

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Chapter 2 Designing an RQ Experiment Selecting the PCR Method Both methods give equivalent results for relative quantification experiments. Which method to use depends on the • Type of chemistry you will be using to detect PCR products – Singleplex PCR can use either SYBR ® Green or TaqMan reagent-based chemistry. Multiplex PCR can use only TaqMan chemistry. • Amount of time you want to spend optimizing and validating your experiment – Amplifying target sequences and endogenous controls in separate reactions (singleplex PCR) requires less optimization and validation than multiplex PCR. Among the factors to consider when doing multiplex PCR are primer limitation, the relative abundance of the target and reference sequences (the endogenous control must be more abundant than the targets), and the number of targets in the study. IMPORTANT! As the number of gene targets increases, the singleplex format is typically more effective than the multiplex format, because less optimization is required. • Requirement for high throughput performance – Running multiple reactions in the same tube in a multiplex experiment increases throughput and reduces the effects of pipetting errors. For more information about multiplex and singleplex PCR, refer to the Sequence Detection Systems Chemistry Guide (PN 430019). Example The singleplex PCR method was used in the sample experiment for the following reasons: • Large number of targets to be amplified (23 genes, plus one endogenous control) • No requirement for optimization and validation for singleplex experiments DRAFT September 26, 2003 3:14 pm, C2_Design.fm Notes 8 Relative Quantification Getting Started Guide for 7000 v1.1
Specifying the Components of an RQ Experiment Specifying the Components of an RQ Experiment RQ experiments require: • A target – The nucleic acid sequence that you are studying. • A calibrator – The sample used as the basis for comparative results. • An endogenous control – A gene present at a consistent expression level in all experimental samples. By using an endogenous control as an active reference, you can normalize quantification of a cDNA target for differences in the amount of cDNA added to each reaction. Note that – Each sample type (for example, each tissue in a study comparing multiple tissues) requires an endogenous control. – If samples are spread across multiple plates, each plate must have an endogenous control. Typically, housekeeping genes such as β-actin, glyceraldehyde-3-phosphate (GAPDH), and ribosomal RNA (rRNA), are used as endogenous controls. • Replicate wells – For relative quantification studies, Applied Biosystems recommends the use of three or more replicate reactions per sample and endogenous control to ensure statistical significance. Replicates allow you to preserve data and remove outliers. 2 For more information about these concepts, refer to the Sequence Detection Systems Chemistry Guide. DRAFT September 26, 2003 3:14 pm, C2_Design.fm Notes Relative Quantification Getting Started Guide for 7000 v1.1 9
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 25: Primer Extended on mRNA 5′ 3′ 5
Page 29 and 30: Selecting the Sequence Detection Ch
Page 31 and 32: 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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