Quality and Quantity Assessment of Nucleic Acids and Proteins · Spectrophotometric Quantification...
Transcript of Quality and Quantity Assessment of Nucleic Acids and Proteins · Spectrophotometric Quantification...
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Quality and Quantity Assessment
of Nucleic Acids and Proteins
Ebru Serinsöz Linke, MD/PhD
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• Quantification of nucleic acids
• Quality assessment of nucleic acids
• Quantification of proteins
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• The amount and quality of molecules (nucleic
acids or proteins):
– Reproducibility
– Accuracy
– Efficiency
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Quantification of Nucleic Acids
• Three quantification methods in common use:
– Spectrophotometric measurement
(UV spectrometry)
– Fluorescent dye (Fluorometry) based measurement
– Real-time amplification (Absolute quantification)
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Spectrophotometric Quantification
• Measurement of light
intensity at different
wavelenghts
• Transmittance: the
amount of light that
passes completely
through the sample
• Absorbance:
measurement of light
that is absorbed by the
sample
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Spectrophotometric Quantification
• Bases in RNA/DNA absorb UV at 250-265nm
• Heterocyclic rings
• Measurements at A260nm, A280nm, A230nm
• Concentration estimation
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Spectrophotometric Quantification
• A260nm:
• Lambert-Beer Law: Cµg/µl = A x dilution factor x
• : molar extinction coefficient – physical constant
– Unique
– Amount of absorbance at 260nm of 1M nucleic acid solution
measured in a 1cm path-length cuvette.
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Spectrophotometric Quantification
• A230 and A280 readings
– A260/A280
– A260/A230
• A260: DNA/RNA, Guanidine isothiocyanate
• A270: Phenol, TRIzol
• A280: Proteins
• A230: Phenol, TRIzol, Guanidine HCL
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Spectrophotometric Quantification
Low A260/A280 ratio
• Residual phenol or other
reagent associated with the
extraction protocol
• A very low concentration
(> 10 ng/ul) of nucleic acid
High 260/280 ratio
• RNA/DNA contamination
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A260/A280: 1.8 for DNA, 2.0 for RNA
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Spectrophotometric Quantification
Low A260/A230 ratio :
• Carbohydrate carryover (often a
problem with plants).
• Residual phenol from nucleic
acid extraction.
• Residual guanidine
(often used in column based kits)
• Glycogen used for precipitation.
High A260/A230 ratio
• Making a Blank measurement
on a dirty pedestal
• Using an inappropriate
solution for the Blank
measurement.
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• A260/A230nm ratio 2.0-2.2
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Factors Affecting Absorbance
• A260/A280 ratio:
–pH
– ionic strength
• Water often has an acidic pH
• Buffered solution (Tris-EDTA at pH 8.0)
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Conventional Spectrophotometers
• Conventional
spectrophotometers:
– Requires sample dilution
– Low sensitivity (lower
limit 0.5-1µg nucleic
acid)
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NANOSPECTROPHOTOMETRY
• Miniaturization of UV
spectrophotometers:
–Rapid
–Direct quantification
of nucleic acids in
microvolumes
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NANOSPECTROPHOTOMETRY-
NanoDrop
• Sample retension system
• Inherent surface tension
of liquids
• Microvolume samples
(0.5-2µl)
• Liquid column
Vertical optical path
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NANOSPECTROPHOTOMETRY
• Vertical path length
– Automatically changed
– Shorter path length higher concentration of sample
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NANOSPECTROPHOTOMETRY
• Benefits:
1. Small sample volume at 0.5-2µl
2. Large dynamic range (2ng/µl-3700ng/µl)
3. Cuvette free operation
4. Short measurement time
5. High accuracy and good reproducibility.
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NANOSPECTROPHOTOMETRY
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NANOSPECTROPHOTOMETRY
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Quantification with a Fluorescent Dye
• DNA/RNA intercalating dyes
• Measurement of fluorescence
• ~1000 times more sensitive than UV absorbance
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Quantification with a Fluorescent Dye
• Ethidium Bromide (EtBr)
• SYBR Green
• Hoechst 33258
• PicoGreen
• RiboGreen
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Quantification with a Fluorescent Dye
EtBr:
• PCR products, gDNA
• Band intensity calculation
• Comparison to known
reference
• Agarose and PAGE
• Not precise, relative
SYBR Green I:
• Highly sensitive
– 25-100times more than
EtBr
• ssDNA, dsDNA
• Agarose and PAGE
• Less mutagenic
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Quantification with a Fluorescent Dye
• Fluorometer
• Hoechst 33258 (DNA): – Binds to A-T bp in dsDNA
– Emission Max. At 460nm
• PicoGreen (DNA)
• RiboGreen (RNA)
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Emission max. at 530nm
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Quantification with a Fluorescent Dye
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TBS-380 Fluorometer 3800: 2mL or 50µl with minicell adapter NanoDrop 3300
Fluorospectrometer
: 2µl
Qubit: 2ooµl
Aquaflour: 2mL
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Quantification with a Fluorescent Dye:
Qubit
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Quantification with a Fluorescent Dye
The Qubit® 2.0 Fluorometer
displays the standard curve
after completion of the
calibration.
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Quantification with a Fluorescent Dye
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Quantification by Real-Time PCR
• DNA, RNA (cDNA)
• Absolute quantification
• Serially diluted standards
Standard curve
• Determination of
concentration of unknowns
based on Ct (Threshold
cycle) values
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Quality Assessment of Nucleic
Acids
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Quality Assessment of Nucleic Acids
• Level of degradation
• Assay amenability (FFPE tissues)
• Method: nature of nucleic acid
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Methods of Quality Assessment of
Nucleic Acids
DNA
1. Agarose gel
electrophoresis
2. PCR amplification of
fragments with
increasing length
RNA
1. Denaturing gel
electrophoresis
2. RT-PCR amplification
of mRNA fragments of
increasing length
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DNA and RNA
1. Agilent 2100 Bioanalyzer
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Agarose gel electrophoresis (DNA)
• gDNA on agarose gel
• EtBr/SYBR Green
staining
• MW marker
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PCR Amplification of Fragments with
Increasing Length (DNA)
• Archival tissues
• Spesmen control size
ladder
• Multiplex PCR
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600bp
400bp
200bp
100bp
MW
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Denaturing Agarose Gel Electrophoresis
(RNA)
• Most common method of integrity
assessment :
• Secondary structure of RNA
altered migration pattern
– Electrophoresis buffer :Formaldehyde and
MOPs (3-[N-Morpholino]-propanesulfonic
acid)
– Loading buffer: Glyoxal
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RT-PCR Amplification of mRNA
Fragments of Increasing Length
• mRNA integrity assessment
• Housekeeping gene
• cDNA subjected to amplification
• Fragments with increasing length
• Independent from rRNA integrity
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Quality Assessment with Capillary
Microchip Electrophoresis:
• LabChip systems (Caliper)
• MCE-202 MultiNA microchip electrophoresis
system (Shimadzu)
• P/ACE MDQ (Beckman Coulter)
• 2100 BioAnalyzer (Agilent ): microfluidics, capillary
electrophoresis, fluorescent detection
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Agilent 2100 BioAnalyzer
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Agilent 2100 BioAnalyzer
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28S/18S ratios 2
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The RNA Integrity Number (RIN)
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• 28S/18S rRNA ratio is reasonable but not ideal!!!
• Nondenaturing conditions
• Software algorithm for the the entire
electrophoretic trace
• Estimation of the integrity of total RNA samples.
• Numbering system 1-10
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RIN
RIN 1 : most degraded
RIN 10: most intact
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RIN
• Downstream application
– RT-PCR vs Microarray
• RINs > 7-8 work well for most experiments.
• RINs < 7 require extra validation studies
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Quantification of Proteins
• Different methods:
– Accuracy required
– Amount and purity of protein
• Spectrophotometric assays:
– UV Absorbance methods
– Colorimetric and fluorescent-based detection
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Quantification of Proteins
• Assay selection criteria:
1. Sample volume (microplate assay vs cuvette-based)
2. Sample recovery (UV spectroscopy)
3. Throughput (microplate compatible rapid assay)
4. Robustness (repeatability)
5. Chemical modifications (Covalent modifications!)
6. Protein aggregation (solubility of the protein)
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Quantification of Proteins
• UV absorbance: quantitation of purified protein
– Proteins that contain Trp, Tyr residues
– Cys-Cys disulphide bonds
• Colorimetric assays: uncharacterized protein
solutions and cell lysate
• Bradford
• BCA
• Lowry
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Quantification of Proteins: UV
Absorption Spectroscopy
• UV Absorption at 280nm
– Range 20-3000µg
– Aromatic aa (tyrosine and tryptophan)
– Molar extinction coefficient
• Beer-Lambert Law: A = am x C x l
– Protein standard
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Quantification of Proteins: UV
Absorption Spectroscopy
• UV Absorption at 205nm
– Range 1-100µg
– Arsorption of photons by peptide bonds
• Molar extinction coefficient at A205
• Protein Standard
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UV Absorption Spectroscopy
• NanoDrop 1000, NanoDrop 8000 A280
modules
• Concentration of purified protein samples
• 1µl sample
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Dye-Based Assays: Bradford Assay
Bradford (Coomassie Blue) Assay (1-50µg):
• Binding of Coomassie Brillant Blue mainly to
Tryptophan and tyrosine residues at acidic pH
• Shift in the absorbance of acidic CB solution
from 465nm to 595nm
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Bradford Assay
48 BMP, Gebze, 19.09.2013
+
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Dye-Based Assays: Lowry Assay
• Lowry (Alkaline Copper Reduction) Assay (5-100µg):
– Two-step procedure
– Reduction of Cu by proteins in alkaline solutions
– Reduction of Folin reagent (a mixture of phosphotungstic
acid and phosphomolybdic acid)
– A blue color is formed with absorbance max. at 750nm
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Dye-Based Assays: Bicinchoninic
Assay
Bicinchoninic (BCA) Assay (0.2-50µg)
• Bicinchoninic acid (replacement of Folin’s
reagent)
• Improved sensitivity
• Tolerance to interfering substances
• Intense purple complex (562nm)
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Protein analysis with the
Agilent 2100 Bioanalyzer
• Microvolume analysis
• Different assays
– For protein analysis in the low molecular weight range
– General protein analysis up to 230 kDa
– Picogram sensitivity
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Quality Assessment of Proteins
1. Composition-Based and activity-based analysis
2. Electrophoretic methods (SDS gel electrophoresis)
3. Chromatographic methods
I. Gel filtration Chromatography
II. Reversed phase HPLC
4. Sedimentation velocity methods
5. Mass spectrometry methods
6. Light scattering methods
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