Introduction; QA and QC in the Molecular Lab
- Recall proper nomenclature for gene names and mutations (aa and nucleic acids)
- Recognize proper specimen handling, accession, and PPE in the molecular lab
● Specimen Handling/Accession
○ Errors
■ Preanalytical - occurs prior to sample analysis
● Patient Identification
● Specimen handling or contamination
● Test order
○ QA vs. QC (Quality Management)
■ Quality Assurance - ensures you are performing a process correctly
● Process-oriented
■ Quality Control - ensures that a product performs appropriately
● Product-oriented
● Importance of PPE
○ ALL specimens are potentially infectious.
○ Standard precautions when handling - gloves, lab coat protect:
■ Lab personnel
■ Nucleic acids from nuclease degradation
● Especially RNA by RNAses
- Describe controls used in the molecular lab
● Samples that are known type or amount (range) that are treated like and run with
patient specimens.
○ Qualitative methods vs. Quantitative methods
○ Negative control/Blank
■ Avoid false positives
, ○ Positive control
■ Contains a low concentration of target
○ Sensitivity control
■ The lower limit for the analytical range
○ Internal control
■ Avoid false negatives
- Recognize optimal conditions for holding and storing specimens and nucleic acids
● DNA - refrigerate
● RNA - freeze
● Collection: (whole blood is best)
○ EDTA (Lavender)
○ Acid citrate dextrose (yellow)
○ RNA tube
Nucleic Acid Extraction Methods
- Recognize factors to consider when choosing an extraction method and means to achieve
high-quality samples
● Processing speed
● Ease of use
● Yield of DNA or RNA
● Quality of DNA and RNA prepared (amplification performance)
● Shelf life/storage conditions
● Quality assurance criteria
● Cost of preparation
- List the basic steps of obtaining a nucleic acid prep
1. Extract
a. Separate WBCs from RBCs, if necessary
b. Lyse WBCs or other nucleated cells
i. Detergents
ii. Heat
iii. Mechanical
2. Isolate
a. Denature/digest proteins
b. Separate contaminants
i. proteins, heme, RNA or DNA
3. Purify
a. Precipitate DNA
i. Isopropanol
ii. Ethanol
, b. Resuspend DNA in final buffer
i. TE
- Compare and contrast organic, inorganic, and solid-phase methods of DNA extraction
including advantages and disadvantages of each (if applicable)
● Organic
○ Uses organic chemicals: phenol, chloroform
■ Combination of ↓pH, ↑salt, organic mix of phenol & chloroform
○ Advantages
■ ↑purity, ↑recovery
○ Disadvantages
■ Slow, labor-intensive, toxic:
● fume-hood & proper disposal of hazardous materials required
● Inorganic
○ Uses inorganic chemicals, detergents, ethylenediaminetetraacetic acid
(EDTA), acetic acid, salt (salting out, spooling)
■ "Salting out" - ↓pH, ↑salt selectively precipitates proteins
■ DNA in aqueous solution is precipitated with isopropanol
○ Advantages
■ Fast, easy
■ Non-toxic
■ Produces good-quality DNA
○ Disadvantages
■ Yield and quality not as good as phenol/chloroform method
● Solid phase
○ DNA is immobilized on a solid support, beads, or columns
■ Solid support columns: bind DNA to separate from other
contaminants
● Glass or Silica binding, Anion-exchange chromatography,
Magnetic bead binding, etc.
■ Nucleic acid binding to silica or magnetic beads is the basis for
many automated extraction systems
○ Fast, easy method; No precipitation required
- Describe how to ensure a high-quality RNA specimen
● RNA is labile - avoid sample degradation
● Lab must have RNAse free area
● Use sterile, disposable plastic materials (tubes, filter tips) marked "For RNA Use
Only"
- Distinguish between the isolation of total RNA with that of mRNA
● RNA extraction methods include organic and solid-phase methods
● mRNA extraction by using immobilized polyT or polyU oligomers
- Recall proper nomenclature for gene names and mutations (aa and nucleic acids)
- Recognize proper specimen handling, accession, and PPE in the molecular lab
● Specimen Handling/Accession
○ Errors
■ Preanalytical - occurs prior to sample analysis
● Patient Identification
● Specimen handling or contamination
● Test order
○ QA vs. QC (Quality Management)
■ Quality Assurance - ensures you are performing a process correctly
● Process-oriented
■ Quality Control - ensures that a product performs appropriately
● Product-oriented
● Importance of PPE
○ ALL specimens are potentially infectious.
○ Standard precautions when handling - gloves, lab coat protect:
■ Lab personnel
■ Nucleic acids from nuclease degradation
● Especially RNA by RNAses
- Describe controls used in the molecular lab
● Samples that are known type or amount (range) that are treated like and run with
patient specimens.
○ Qualitative methods vs. Quantitative methods
○ Negative control/Blank
■ Avoid false positives
, ○ Positive control
■ Contains a low concentration of target
○ Sensitivity control
■ The lower limit for the analytical range
○ Internal control
■ Avoid false negatives
- Recognize optimal conditions for holding and storing specimens and nucleic acids
● DNA - refrigerate
● RNA - freeze
● Collection: (whole blood is best)
○ EDTA (Lavender)
○ Acid citrate dextrose (yellow)
○ RNA tube
Nucleic Acid Extraction Methods
- Recognize factors to consider when choosing an extraction method and means to achieve
high-quality samples
● Processing speed
● Ease of use
● Yield of DNA or RNA
● Quality of DNA and RNA prepared (amplification performance)
● Shelf life/storage conditions
● Quality assurance criteria
● Cost of preparation
- List the basic steps of obtaining a nucleic acid prep
1. Extract
a. Separate WBCs from RBCs, if necessary
b. Lyse WBCs or other nucleated cells
i. Detergents
ii. Heat
iii. Mechanical
2. Isolate
a. Denature/digest proteins
b. Separate contaminants
i. proteins, heme, RNA or DNA
3. Purify
a. Precipitate DNA
i. Isopropanol
ii. Ethanol
, b. Resuspend DNA in final buffer
i. TE
- Compare and contrast organic, inorganic, and solid-phase methods of DNA extraction
including advantages and disadvantages of each (if applicable)
● Organic
○ Uses organic chemicals: phenol, chloroform
■ Combination of ↓pH, ↑salt, organic mix of phenol & chloroform
○ Advantages
■ ↑purity, ↑recovery
○ Disadvantages
■ Slow, labor-intensive, toxic:
● fume-hood & proper disposal of hazardous materials required
● Inorganic
○ Uses inorganic chemicals, detergents, ethylenediaminetetraacetic acid
(EDTA), acetic acid, salt (salting out, spooling)
■ "Salting out" - ↓pH, ↑salt selectively precipitates proteins
■ DNA in aqueous solution is precipitated with isopropanol
○ Advantages
■ Fast, easy
■ Non-toxic
■ Produces good-quality DNA
○ Disadvantages
■ Yield and quality not as good as phenol/chloroform method
● Solid phase
○ DNA is immobilized on a solid support, beads, or columns
■ Solid support columns: bind DNA to separate from other
contaminants
● Glass or Silica binding, Anion-exchange chromatography,
Magnetic bead binding, etc.
■ Nucleic acid binding to silica or magnetic beads is the basis for
many automated extraction systems
○ Fast, easy method; No precipitation required
- Describe how to ensure a high-quality RNA specimen
● RNA is labile - avoid sample degradation
● Lab must have RNAse free area
● Use sterile, disposable plastic materials (tubes, filter tips) marked "For RNA Use
Only"
- Distinguish between the isolation of total RNA with that of mRNA
● RNA extraction methods include organic and solid-phase methods
● mRNA extraction by using immobilized polyT or polyU oligomers
