MIDTERM LEC 2: RNA ISOLATION

Question 1

RAPID EXTRACTION METHODS

Answer 1

• PCR
• Rapid Lysis methods
• DNA extraction/storage cards
• Chelex

Question 2

o Easy to use systems for collection preservation, & long – term storage of nucleic acids at room temperature (mainly for storage)

Answer 2

DNA extraction/storage cards

Question 3

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.

Answer 3

 Chelex

Question 4

to separate 700 – 2,600 x g pellets

Answer 4

LOW – SPEED CENTRIFUGATION

Question 5

(10,000 – 16, 000 x g) pellet contains
mitochondria and lysed with detergents
(SDS); lysate will be added with proteinase

Answer 5

HIGH – SPEED CENTRIFUGATION

Question 6

RNAses inhibitors

Answer 6

• diethyl pyrocarbonate (DEPC)
• vanadyl-ribonucleoside complexes
• macaloid clays

Question 7

Most abundant (80% - 90%) RNA
in all cells

Answer 7

rRNA (Ribosomal RNAs)

Question 8

Next most abundant RNA fraction (2.5% to 5%)

Answer 8

mRNA (Messenger RNAs)

Question 9

RNA ISOLATION

Answer 9

• 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

Question 10

To stabilize RNA immediately upon draw

Answer 10

Specialized collection tubes

Question 11

Specialized collection tubes?

Answer 11

Tempus™ Blood RNA Tubes or
Pax - gene Blood RNA Tubes

Question 12

II. Preparation of Specimen Material

LYSED BY OSMOSIS or separated from WBCs by centrifugation

Answer 12

Reticulocytes in blood & bone marrow samples

Question 13

Kept frozen in liquid nitrogen / immersed in buffer that will inactivate intracellular RNAses

Answer 13

TISSUE

Question 14

Isolated by chemical lysis by grinding in liquid nitrogen

Answer 14

BACTERIAL & FUNGAL RNA

Question 15

Isolated directly from the serum, plasma culture medium, or other cell-free fluids by means of specially formulated spin columns or beads

Answer 15

VIRAL RNA

Question 16

III. RNA Isolation Chemistries
ORGANIC ISOLATION
Acid phenol: chloroform: isoamyl alcohol
RATIO OF SOLUTION to efficiently extracts RNA

Answer 16

25:24:1

Question 17

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

Answer 17

ORGANIC SOLUTION

Question 18

 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

Answer 18

SOLID - PHASE ISOLATION

Question 19

1 million eukaryotic cells or 10-50 mg of tissue = ???

Answer 19

10 μg of RNA

Question 20

The quality of RNA from fixed, paraffin-embedded tissue will depend on the _____________ and HANDLING OF THE SPECIMEN BEFORE FIXATION

Answer 20

fixation process

Question 21

RNA often required for analysis:

Answer 21

mRNA A (2.5% - 5% of the total RNA yield)

Question 22

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

Answer 22

ELECTROPHORESIS

Question 23

Technique that uses light absorption to measure the concentration of an analyte in a solution

Answer 23

SPECTROPHOTOMETRY

Question 24

Measurement of emitted fluorescent light

Answer 24

FLUOROMETRY

Question 25

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

Answer 25

MICROFLUIDICS

Question 26

Fluorescent dyes used in ELECTROPHORESIS

Answer 26

ETHIDIUM BROMIDE SYBR GREEN I SILVER STAIN

Question 27

Nucleic acids absorb light at ___ nm through adenine residues

Answer 27

260

Question 28

Beer-Lambert Law

Answer 28

Absorbance is directly proportional to the concentration of the nucleic acid in the sample FORMULA: A= ∈bc

Question 29

To determine concentration:

Answer 29

Spectrophotometer reading in absorbance units × appropriate conversion factor

Question 30

If the DNA / RNA preparation require dilution before spectrophotometry, to determine concentration:

Answer 30

Absorbance reading x conversion factor x dilution factor

Question 31

Absorbance of DNA at 260 nm

Answer 31

1.6 to 2.00

Question 32

Absorbance of RNA at 260 nm

Answer 32

= 2.0 to 2.3

Question 33

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

Answer 33

contaminated

Question 34

Most likely contaminant

Answer 34

PROTEINS (absorbs light at 260 nm through the aromatic tryptophan & tyrosine residues

Question 35

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

Answer 35

UV Spectrophotometry

Question 36

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

Answer 36

NanoDrop Spectrophotometry

Question 37

 Measure up to 200 ng DNA/mL  Measures fluorescence related to DNA concentration in association with DNA-specific fluorescent dyes

Answer 37

Fluorometry (Fluorescent Spectroscopy)

Question 38

Fluorometry (Fluorescent Spectroscopy) Early methods:

Answer 38

3,5-diaminobenzoic acid 2HCl (DABA)

Question 39

Modern methods:

Answer 39

DNA-specific dye Hoechst 33258

Question 40

Other DNA-specific dyes - detection down to 25 pg/mL concentrations

Answer 40

PicoGreen

Question 41

Other DNA-specific dyes - detection down to 100 pg/mL concentrations

Answer 41

OilGreen

Question 42

Specific dye use in RNA?

Answer 42

SybrGreen II RNA gel stain

Question 43

 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

Answer 43

Microfluidics