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MOL. BIO LEC > MIDTERM LEC 2: RNA ISOLATION > Flashcards

MIDTERM LEC 2: RNA ISOLATION Flashcards

1
Q

RAPID EXTRACTION METHODS

A
  • PCR
  • Rapid Lysis methods
  • DNA extraction/storage cards
  • Chelex
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2
Q

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

A

DNA extraction/storage cards

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3
Q

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.

A

 Chelex

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4
Q

to separate 700 – 2,600 x g pellets

A

LOW – SPEED CENTRIFUGATION

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5
Q

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

A

HIGH – SPEED CENTRIFUGATION

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6
Q

RNAses inhibitors

A
  • diethyl pyrocarbonate (DEPC)
  • vanadyl-ribonucleoside complexes
  • macaloid clays
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7
Q

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

A

rRNA (Ribosomal RNAs)

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8
Q

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

A

mRNA (Messenger RNAs)

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9
Q

RNA ISOLATION

A
  • 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
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10
Q

To stabilize RNA immediately upon draw

A

Specialized collection tubes

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11
Q

Specialized collection tubes?

A

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

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12
Q

II. Preparation of Specimen Material

LYSED BY OSMOSIS or separated from WBCs by centrifugation

A

Reticulocytes in blood & bone marrow samples

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13
Q

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

A

TISSUE

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14
Q

Isolated by chemical lysis by grinding in liquid nitrogen

A

BACTERIAL & FUNGAL RNA

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15
Q

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

A

VIRAL RNA

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16
Q

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

A

25:24:1

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17
Q

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

A

ORGANIC SOLUTION

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18
Q

 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

A

SOLID - PHASE ISOLATION

19
Q

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

A

10 μg of RNA

20
Q

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

A

fixation process

21
Q

RNA often required for analysis:

A

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

22
Q

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

A

ELECTROPHORESIS

23
Q

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

A

SPECTROPHOTOMETRY

24
Q

Measurement of emitted fluorescent light

A

FLUOROMETRY

25
Q

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

A

MICROFLUIDICS

26
Q

Fluorescent dyes used in ELECTROPHORESIS

A

ETHIDIUM BROMIDE
SYBR GREEN I
SILVER STAIN

27
Q

Nucleic acids absorb light at ___ nm through adenine residues

A

260

28
Q

Beer-Lambert Law

A

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

29
Q

To determine concentration:

A

Spectrophotometer reading in absorbance units × appropriate conversion factor

30
Q

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

A

Absorbance reading x conversion factor x dilution factor

31
Q

Absorbance of DNA at 260 nm

A

1.6 to 2.00

32
Q

Absorbance of RNA at 260 nm

A

= 2.0 to 2.3

33
Q

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

A

contaminated

34
Q

Most likely contaminant

A

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

35
Q

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

A

UV Spectrophotometry

36
Q

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

A

NanoDrop Spectrophotometry

37
Q

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

A

Fluorometry (Fluorescent Spectroscopy)

38
Q

Fluorometry (Fluorescent Spectroscopy)
Early methods:

A

3,5-diaminobenzoic acid 2HCl (DABA)

39
Q

Modern methods:

A

DNA-specific dye Hoechst 33258

40
Q

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

A

PicoGreen

41
Q

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

A

OilGreen

42
Q

Specific dye use in RNA?

A

SybrGreen II RNA gel stain

43
Q

 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

A

Microfluidics

MOL. BIO LEC (14 decks)

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