MIDTERM LEC 2: RNA ISOLATION Flashcards
RAPID EXTRACTION METHODS
- PCR
- Rapid Lysis methods
- DNA extraction/storage cards
- Chelex
o Easy to use systems for collection preservation, & long – term storage of nucleic acids at room temperature (mainly for storage)
DNA extraction/storage cards
o Cation-chelating resin that can be used for simple extraction of DNA from minimal samples.
o 10% Chelex resin beads is mixed with the specimen & the cells are lysed by boiling.
Chelex
to separate 700 – 2,600 x g pellets
LOW – SPEED CENTRIFUGATION
(10,000 – 16, 000 x g) pellet contains
mitochondria and lysed with detergents
(SDS); lysate will be added with proteinase
HIGH – SPEED CENTRIFUGATION
RNAses inhibitors
- diethyl pyrocarbonate (DEPC)
- vanadyl-ribonucleoside complexes
- macaloid clays
Most abundant (80% - 90%) RNA
in all cells
rRNA (Ribosomal RNAs)
Next most abundant RNA fraction (2.5% to 5%)
mRNA (Messenger RNAs)
RNA ISOLATION
- SPECIMEN COLLECTION
- CHEMISTRIES
o Organic isolation
o Solid – phase isolation
o Proteolytic lysis of fixed materials
o Rapid extraction methods
o Isolation of PolyA (messenger) RNA
To stabilize RNA immediately upon draw
Specialized collection tubes
Specialized collection tubes?
Tempus™ Blood RNA Tubes or
Pax - gene Blood RNA Tubes
II. Preparation of Specimen Material
LYSED BY OSMOSIS or separated from WBCs by centrifugation
Reticulocytes in blood & bone marrow samples
Kept frozen in liquid nitrogen / immersed in buffer that will inactivate intracellular RNAses
TISSUE
Isolated by chemical lysis by grinding in liquid nitrogen
BACTERIAL & FUNGAL RNA
Isolated directly from the serum, plasma culture medium, or other cell-free fluids by means of specially formulated spin columns or beads
VIRAL RNA
III. RNA Isolation Chemistries
ORGANIC ISOLATION
Acid phenol: chloroform: isoamyl alcohol
RATIO OF SOLUTION to efficiently extracts RNA
25:24:1
Cell Lysis
o Detergent or phenol in the presence of high salt (0.2 to 0.5 M NaCl) or RNAse inhibitors
o GUANIDINE ISOTHICYANATE (GITC) can also be used
o Strong reducing agents (2 MERCAPTOETHANOL) may also be added
ORGANIC SOLUTION
Begins with similar steps as described for organic extraction
The strong denaturing buffer conditions must be adjusted before application of the lysate to the column
SOLID - PHASE ISOLATION
1 million eukaryotic cells or 10-50 mg of tissue = ???
10 μg of RNA
The quality of RNA from fixed, paraffin-embedded tissue will depend on the _____________ and HANDLING OF THE SPECIMEN BEFORE FIXATION
fixation process
RNA often required for analysis:
mRNA A (2.5% - 5% of the total RNA yield)
Measurement of Nucleic Acid Quality & Quantity
- DNA and RNA can be analyzed for QUALITY (detection & size analysis by resolving an aliquot of the isolated
sample on an agarose gel)
- Separation of particles through a solution or matrix under the force of an electric current
ELECTROPHORESIS
Technique that uses light absorption to measure the concentration of an analyte in a solution
SPECTROPHOTOMETRY
Measurement of emitted fluorescent light
FLUOROMETRY
Science & technology of systems that
process or manipulate small amounts of fluid (10-9 to 10-18L), using channels measuring from tens to hundreds of micrometers
MICROFLUIDICS
Fluorescent dyes used in ELECTROPHORESIS
ETHIDIUM BROMIDE
SYBR GREEN I
SILVER STAIN
Nucleic acids absorb light at ___ nm through adenine residues
260
Beer-Lambert Law
Absorbance is directly proportional to the concentration of the nucleic acid in the sample
FORMULA: A= ∈bc
To determine concentration:
Spectrophotometer reading in absorbance units × appropriate conversion factor
If the DNA / RNA preparation require dilution before spectrophotometry, to determine concentration:
Absorbance reading x conversion factor x dilution factor
Absorbance of DNA at 260 nm
1.6 to 2.00
Absorbance of RNA at 260 nm
= 2.0 to 2.3
If the 260 nm / 280 nm ratio is <1.6, the nucleic acid preparation may be
________ with unacceptable amounts of protein & NOT OF SUFFICIENT PURITY FOR USE
contaminated
Most likely contaminant
PROTEINS (absorbs light at 260
nm through the aromatic tryptophan & tyrosine residues
o Standard nucleic acid quantitation
o Nucleic acid sample is placed into quartz cuvette,
which is then placed inside the UV spectrophotometer
Light source: UV (260nm wavelength)
o UV light passed through the sample at a specified path length, & the absorbance of the sample at specific wavelengths is measured
o Does not require additional reagents / incubation time
UV Spectrophotometry
o Similar in principle with the previous, but has many additional capabilities:
o Functions by combining filter optic technology & natural surface tension properties
o Accompanied by special software to enable analysis of signal from small quantities of sample
o Displays the entire absorbance spectrum of the sample in graphical form – ALLOWS DETECTION OF CONTAMINANTS
o Capable of determining a WIDE RANGE OF SAMPLE CONCENTRATIONS w/o requiring serial dilutions
NanoDrop Spectrophotometry
Measure up to 200 ng DNA/mL
Measures fluorescence related to DNA concentration
in association with DNA-specific fluorescent dyes
Fluorometry (Fluorescent Spectroscopy)
Fluorometry (Fluorescent Spectroscopy)
Early methods:
3,5-diaminobenzoic acid 2HCl (DABA)
Modern methods:
DNA-specific dye Hoechst 33258
Other DNA-specific dyes
- detection down to 25 pg/mL
concentrations
PicoGreen
Other DNA-specific dyes
- detection down to 100 pg/mL
concentrations
OilGreen
Specific dye use in RNA?
SybrGreen II RNA gel stain
Sample is applied to a multiwell chip & then moves through microchannels across a detector
Instrument software generates image in electropherogram (peak) or gel (band) configurations
RNA integrity number: quantification estimate for RNA, determined as a standard measure of RNA integrity
Uses a minimal volume of sample (as low as 1 μL) & can test multiple samples simultaneously
Useful for analysis of studies on small TNAs (microRNAs) in eukaryotes & gene expression in bacteria
Microfluidics
