Biotechnology Engineering

1. ANALYSIS OF RNA AND
NORTHERN HYBRIDIZATION
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Lecture- 18

2. Storage of RNA
• RNA precipitate is dissolved in deionized formamide and
stored at -20°C
• RNA precipitate is dissolved in aqueous buffer, which
minimizes hydrolysis of RNA.
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3. Analysis of RNA Preparation
• Denaturing agarose gel electrophoresis
• UV spectrophotometry
• Colorimetric analysis by ornicol method
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4. Northern blotting and Northern
hybridization
•Technique for detecting specific RNAs
separated by electrophoresis by
hybridization to a labeled nucleic acid
probe
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5. The flow chart of Northern hybridization
Prepare RNA samples and run RNA gel
Northern transfer
Probe preparation
Prehybridization
Hybridization
Post-hybridization washing
Signal detection
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6. Preparation of agarose/formaldehyde gel
•E.g. Prepare a 350 ml 1.2%
agarose/formaldehyde gel
 4.2 g agarose in 304.5 ml water.
Microwave, then cool to 60C. Add
35 ml 10x MOPS running buffer and
10.5 ml 37% formaldehyde
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7. Preparation of an RNA sample
• Prepare a premix:
• 5 l of 10x MOPS running buffer
• 8.75 l of 37% formaldehyde
• 25 l of formamide.
• Prepare RNA sample:
• 38.75 l of premix
• RNA (0.5 to 10 g)*
• water to 50 l
• *If the mRNA species of interest makes up a relatively high
percentage of the mRNA in the cell (>0.05% of the message), total
cellular RNA can be used. If the mRNA species of interest is relatively
rare, however, it is advisable to use poly(A)+ RNA.
• Incubate 15 min at 55C
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8. Running the RNA gel
•Add 10 l formaldehyde loading buffer
to an RNA sample and load gel. Run
gel at 100 to 120 V for ~3hr.
•Remove gel from the running tank and
rinse several times in water. Place gel
in 10x SSC for 45 min.
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9. Methods for preparation of probes
•Synthesis of uniformly labeled double-
stranded nucleic acid probe
•Preparation of single-stranded probe
•Labeling the 5 and 3 termini of RNA
•Synthesis of RNA probe by in vitro
transcription by bacteriophage DNA-
dependent RNA polymerase
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10. Prehybridization
•Add prehybridization solution and
prehybridize at hybridization
temperature for 2-4 hr
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11. Hybridization
 Remove prehybridization
solution and add
hybridization solution
 Hybridize overnight at
appropriate temperature
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12. Post-hybridization washing
• Wash twice, 15 min each, in 1x SSC, 0.1%
SDS at room temperature
• Wash twice, 15 min each, in 0.25x SSC,
0.1% SDS at hybridization temp
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13. Markers used in gels for RNA
• RNA standards purchased from a
commercial source
• DNA standards purchased from a
commercial source
• Highly abundant rRNAs (28S and 18S)
within the RNA preparations under test
• Tracking dyes
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14. Transfer of RNA from agarose gel to solid
support
• Upward capillary transfer - Overnight transfer of
RNA from gel to membrane in an upward flow of
buffer
• Problems of retaining large RNA molecules in gel
can be overcome :
by using thinnest gel possible
by ensuring that filter papers are saturated with
buffer before transfer
by partial hydrolysis of RNA by alkali
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15. Downward capillary transfer
• Descending transfer does not cause flattening of
agarose gel and results in a faster transfer of
RNA
• Eg: RNA molecules up to 8 kb in size are
transferred with high efficiency within 1 hr at
either neutral or alkaline pH
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16. Membranes used
Unmodified Nylon
• Capacity 200-300 μg
• Size of nucleic acid >50
bp
Charged modified nylon
• 400-500 μg
• Size of nucleic acid >50
bp
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17. An example of Northern blotting
Northern blot
RNA gel
28 S
18 S
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