ISO 20395:2019-08

Biotechnologie - Anforderungen zur Bewertung von Quantifizierungverfahren für Nukleinsäurezielsequenzen - qPCR und dPCR

Englischer Titel: Biotechnology - Requirements for evaluating the performance of quantification methods for nucleic acid target sequences - qPCR and dPCR
Ausgabedatum: 2019-08
Originalsprachen: Englisch
Seiten: 50

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Ausgabedatum: 2019-08
Originalsprachen: Englisch
Seiten: 50

Produktinformationen auf dieser Seite:

Inhaltsverzeichnis
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Inhaltsverzeichnis

Inhalt (en)

Foreword
Introduction
Scope
Normative references
Terms and definitions
Design of measurement procedure
- General
- Quantification method
  - General
  - qPCR determination of nucleic acid concentrations using a calibration curve
  - dPCR determination of copy number concentration using molecular counting
  - Relative quantification by qPCR
  - dPCR determination of ratio between two targets
- Normalization strategy
- Controls
Sample QC — Total nucleic acid quantity, integrity and purity
- General
- Total nucleic acid quantification
  - General
  - Spectrophotometry
  - Fluorometry
  - Assessment of total DNA concentration using qPCR/dPCR
- Nucleic acid integrity
- Nucleic acid purity
Assay design and optimization for quantification of nucleic acid target sequences
- Assay design
  - General
  - Amplicon selection
  - Primer and probe design
  - In silico evaluation of specificity
  - RT-qPCR/RT-dPCR design
- Assay optimization using purified samples
  - General
  - Optimization of fluorescence signal
  - (RT)-qPCR amplification efficiency
  - RT efficiency
  - Specificity
- Method optimization using test samples
  - Effect of PCR inhibitors in sample matrix
  - Presence of nucleic acid contaminants in test sample
  - Validated measurement range
- No template controls
Data quality control (QC) and analysis
- General
- Acceptance criteria
  - qPCR
  - dPCR
- Threshold setting
  - qPCR
  - dPCR
- Data pre-processing
  - qPCR using calibration curve
  - Relative quantification (qPCR)
- Identification of outliers
Nucleic acid quantification measurement method validation
- General
- Precision
- LOQ
- LOD
- Linearity
- Trueness
- Robustness
- Specific considerations for qPCR method validation
  - Repeatability of qPCR- or RT-qPCR
  - Intermediate precision and reproducibility of qPCR- or RT-qPCR
- Specific considerations for dPCR method validation
Nucleic acid quantification measurement traceability and comparability
- Metrological traceability
- Use of reference materials
- Instrument calibration
Measurement uncertainty (MU) in qPCR and dPCR measurements
- General requirements for MU calculations
- qPCR measurement uncertainty
- Ratio-based measurements
- dPCR measurement uncertainty
Reporting
Spectrophotometry (informative)
- General
- Relationship between absorbance and total nucleic acid concentration
- Absorbance ratios and nucleic acid purity
Nucleic acid integrity (informative)
- General
- Techniques for evaluation of DNA integrity
  - Electrophoresis
  - Long range PCR
  - PCR assay with differential size amplicons
- Techniques for evaluation of RNA integrity
  - Electrophoresis
  - RT-qPCR for 5′/3′ mRNA ratio
  - RT-qPCR assay of repetitive elements
  - RT-qPCR assays with different size amplicons
PCR efficiency (informative)
- General
- Experimental design
- Calculations
Measurement uncertainty (informative)
- General
- Sources of uncertainty for whole process (qPCR)
- Sources of uncertainty for dPCR
MIQE and dMIQE checklists (informative)
- General
- MIQE checklist
- dMIQE checklist
Bibliography

ICS

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