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TECHNIQUES IN PLANT VIROLOGY

CIP Training Manual


2.5 DETECTION/Molecular Methods

Section 2.5.3
Preparation of
32
P-Labeled Probes by
RNA
Transcription

This technique for the detection of PSTVd is based on the hybridization of


a highly radioactive molecule of complementary PSTVd RNA, with the
PSTVd present in the sample previously fixed to a nitrocellulose
membrane. The resulting hybrid is detected by autoradiography.

Labeling (RNA transcription)


The transcription system is a process by which the genetic message from
a DNA molecule is transcribed into RNA. This process is limited to
sequences cloned inside specific vectors.
The transcription implies the presence of a promoter (SP6 or T7, for
example) located ahead of the cDNA target in the reaction. The promoter
will be recognized by the corresponding polymerase.

To ensure the synthesis of just the desired sequence, the plasmid is


linearized immediately after the cloned sequence, so the transcription
starts in correspondence with the promoter; continues along the cloned
molecule, and is interrupted at the linearization point.

If one of the nucleotides in the mixture is labeled or radioactive, it will be


included in the synthesis of the new RNA molecule. The degradation of
the plasmid used as a template by means of a DNAse allows recovering
the pure labeled RNA. For labeling, choices are the riboprobe gemini
system (Promega) and the radioactive nucleotide uridine-5’-triphosphate
32
(α P, specific activity: 800 Ci/mmol, concentration: 10 mCi/ml).

Warning: Developing this test implies the use of radioactive material.


Wear gloves through all the steps, being extremely careful when handling
the material. Materials must be disposed of in appropriate containers and
stored following the instructions of the institution responsible for nuclear
32
safety. The half-life of P is 14 days, so the minimum storage time for
radioactivity to vanish is approximately 4 to 5 months.

Mix: in an Eppendorf tube at room temperature (the following reagents)

5 X Transcription buffer 4 µl
DTT 100 mM 2 µl
RNAsin (Promega) 1 µl
GTP 10 mM 1 µl
ATP 10 mM 1 µl
CTP 10 mM 1 µl
Linearized Plasmid (1 µg) 2 µl
α 32P-UTP 10 mCi/ml 2 µl
H2O 5 µl
RNA polymerase SP6 or T7, 1 µl
depending on the promoter
contained in the plasmid

Mix well and centrifuge briefly. Incubate at 38 oC for 120 minutes in a


water bath. Add 1 l RQ1 DNAse (1 unit/ l) and incubate at 37 oC for 20
minutes. Then add 179 l ddH2O and mix. Add 100 l saturated phenol
and 100 l chloroform. Mix in vortex and centrifuge for 2–5 minutes.

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Transfer the supernatant to another Eppendorf tube, add 200 l
chloroform, mix in a vortex, and centrifuge for 2–5 minutes at 14,000
rpm.
Transfer the supernatant to another Eppendorf tube; add 20 l 3 M
Sodium acetate pH 5.2 and 550 l cold ethanol.

Mix and store at –70 OC for a minimum of 30 minutes. Thaw at room


temperature for 5 minutes. Centrifuge for 15 minutes at 14,000 rpm.
Discard supernatant (check at 10 cm distance; the supernatant should
measure 0.5 mR; the pellet from 1 to 1.5 mR at 10X). Turn the Eppendorf
tube upside down to dry the pellet.

Resuspend the pellet in 100 l T10E1 containing 1% 2-mercaptoethanol


and mix thoroughly. Store at –70 0C, until used.

Incorporation and Measuring


Cut 3 MM Whatman filter papers into 2 x 0.5-cm rectangles. Drop 1 l
labeled RNA on the center of the paper and allow to dry completely.
Wash the filter paper as follows:

Twice for 5 minutes in 5% TCA


Once for 5 minutes in 70% ethanol
Once for 5 minutes in absolute ethanol

Allow to dry completely. Place the dry filter paper inside the lead lid used
for transporting the radioactive nucleotide. Place the Geiger counter on
the lid and measure the radioactivity as shown in the table.

Measuring Radioactivity (Calculated by CPM – “Counts per Minute”)


MR Ampl mR x Ampl mR Total x 2500 CPM x 17.8 400,000/ µl/ml x ml B.
= mR Total = CPM = CPM/µl (CPM/µl) Hybridization =
= µl/ml µl Total

Hybridization
Place the membrane in a suitable plastic bag, seal, and cut a corner. For
a 12 x 16-cm membrane, pour 9.6 ml hybridization buffer into the bag,
reseal the bag, and incubate it in a water bath at 55 oC for 10 minutes.

Hybridization buffer x 40 ml Final concentration


Deionized formamide 20 ml 50%
Sodium cacodilate
200 mM 5 ml 25 mM
SDS 20% 0.25 ml 0.125%
Distilled water Up to 40 ml
Denature calf-thymus DNA (Stock: 6 mg/ml), by heating it at 100 oC for 5
minutes, immediately chill on ice. Add 1 ml calf-thymus DNA to the bag,
mix well. Incubate at 55 oC for 10 minutes.

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Add 2.4 ml dextran sulfate (previously warmed to 55 oC, stock 50%),
incubate at 55 oC for 10 more minutes.

To the bag containing the membrane, add the amount of probe


(calculated as mentioned above) necessary to obtain a final
concentration of 400,000 CPM/ml hybridization buffer (for instance, 9.6 +
1 + 2.4 = 13 ml).

Mix very well, and seal the plastic bag carefully to avoid bubbles forming.
Incubate overnight in a water bath at 55 oC for viroids and 45 oC for
viruses.

The next day, cut a corner of the plastic bag and remove the hybridization
solution. Be very careful because the solution is highly radioactive.
Dispose of it in an appropriate container.

Washing

All washing steps should be done in a metal tray, using a rotary shaker.
The washing solutions should be discarded after use as indicated in the
warning above.

Washing buffer 1
0.36 M NaCl (21 g/l)
20 mM Tris-base (2.4 g/l)
37% HCl (1.48 ml/l)
20% SDS (5 ml/l)

Washing buffer 2
0.1X SSC
0.1% SDS

Washing buffer 3
2X SSC

Wash twice in washing buffer 1 for 20 minutes at room temperature;


once in washing buffer 2 for 30 minutes in a water bath at 65 oC; and
twice in washing buffer 3 for 10 minutes at room temperature. Wash
once again for 20 minutes in 100 ml washing buffer 3 with 20 l RNAse
(stock: 10 mg/ml) at room temperature (final concentration: 2 g/ml).
Finally, wash once again in washing buffer 2 for 20 minutes in a water
bath at 50 oC.

After washings, dry the membranes under a warm lamp or at room


temperature.

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Autoradiography
Place the dry membrane in the cassette (work with great care, always
wearing gloves: remember that the membrane is radioactive). Fix the
corners with tape to avoid sliding. Cover the membrane with non-
electrostatic plastic to avoid contaminating the photographic film.

The next steps should take place in a dark room with an appropriate red
light (use a Kodak GBX-2, safelight filter), and handlers should wear
gloves.

a) Place the photographic film (Kodak X-OMAT AR or similar) on the


membrane and cover with intensifying screen.

b) Close the cassette.

c) Store at –70 oC for about 24 hours.

d) Remove the cassette and develop immediately.

e) Remove the plate from the cassette in a dark room, and cut one
corner to identify the original position inside the cassette.

f) Develop for 2 minutes in a metal tray filled with the developer


solution at 22–23 oC, shaking the films gently.

g) Rinse briefly in water.

h) Place the films in the fixer solution for 2 minutes.

i) Rinse again with water and dry the films.

Identify the results by placing the photographic films over the membrane
and matching the position of the spots on the films with the positive spots
on the membrane.

Buffer Preparation
a) 20X SSC (l liter)

175.32 g NaCl (3 M)
88.23 g dehydrated sodium citrate (0.3 M).

Dissolve in 800 ml distilled water. Adjust pH to 7.0 with NaOH. Add


distilled water to make 1 liter. Autoclave if necessary.

b) Deionized Formamide (200 ml)

Mix 200 ml formamide with 7-g mixed-bed, ion-exchange resin


(Bio-Rad AG 501-X8, 20–50 mesh) in a beaker. Stir gently for 1 to
2 hours at room temperature. Filter with Whatman 1 filter paper.
Repeat this step with another 7 g and filter. Divide into aliquots and
store at –20 OC.

c) 20% SDS (100 ml)

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Dissolve 20 g electrophoresis-grade SDS in 90 ml distilled water.
Heat to 68OC to dissolve completely. Adjust pH to 7.2 with
concentrated HCl. Add distilled water to make 100 ml. The mix
does not need to be sterilized.

d) 3 M sodium acetate pH 5.2 (10 ml)

Dissolve 2.46 g CH3COONa (MW 82.03) in 8 ml ddH20. Adjust to


pH 5.2 with CH3COOH. Complete volume up to 10 ml with distilled
water. Autoclave.

e) Sodium Cacodilate buffer (200 mM)

x100 ml Final concentration


Sodium cacodilate 2.72 g 170 mM
Cacodilic acid 0.414 g 30 mM
NaCl 10.4 g 1.8 M
EDTA (Stock: 500 mM pH 8 0) 2 ml 10 mM

Dissolve in sterile distilled water, and make up to final volume.


Wear gloves and mask during preparation because it is very toxic.

f) T10E1 + 1% 2-mercaptoethanol

For 100 ml, dilute 1 ml of 1 M Tris-HCl pH 8.0 and 0.2 ml 500 mM


EDTA, pH 8.0 in dH20, autoclave, and add 1% 2-mercaptoethanol.
Divide into 1-ml aliquots and store at –20 OC.

g) 5% TCA

For 500 ml, dissolve 25 g trichloroacetic acid and 1.42 g Na2HPO4


in sterile distilled water. Stir until completely dissolved and adjust
final volume. The Na2HPO4 concentration will be 0.02 M.

h) Dextran sulfate 50%

Dissolve 50 g dextran sulfate in distilled water; adjust to 100 ml


volume.
Heat in a water bath at 60–70 oC to dissolve completely (the
solution is very dense). Store at –20 oC.

i) EDTA 500 mM pH 8.0

For 100 ml, dissolve 18.6 g EDTA (MW 372.2) in sterile distilled
water. The dissolving process is very slow. Add NaOH in tablets to
adjust pH to 8.0. Add sterile distilled water to adjust final volume to
100 ml.

j) Calf-thymus DNA (6 mg/ml)

Dissolve 1 g calf-thymus DNA in 170 ml sterile distilled water,


shake overnight at 4 oC. (Turn the shaker to high speed, as the
DNA becomes more dense as it dissolves.)

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Pass the dissolved DNA through a syringe 2 or 3 times to break it
up even more.
Divide into 1-ml aliquots in Eppendorf tubes and store at –20 oC.

k) RNAse (10 mg/ml)

In 10 ml bi-distilled H2O, add 50 l 2 M Tris-HCl pH 7.5 (final


concentration: 10 mM) with 40 l 4 M NaCl (final concentration: 15
mM). Weigh 10 mg/ml ribonuclease A (type 1A, Sigma R4875);
shake and dissolve. Place in a water bath at 100 oC for 15 minutes.
Cool to room temperature, divide into aliquots in Eppendorf tubes,
and store at –20 oC.

l) Required solutions

• Labeling: riboprobe gemini II core system kit (Promega, Cat,


No. P-1420), Uridine 5' triphosphate tetratriethyl ammonium
salt -32P-UTP (Nycomed Amersham 1, Cat. No. PB40383)
RQ1 DNase (Promega, cat. No. M6101) 1000 units.
• Measuring incorporation: 5% TCA, 70% ethanol, absolute
ethanol.
• Hybridization: formamide, sodium cacodilate, EDTA, SDS,
calf-thymus DNA (6 mg/ml), dextran sulfate 50%.
• Washing SSC, SDS, RNAse (10 mg/ml).
• Development of photographic films: developer solution, fixer
solution.

Saturated Phenol Preparation


Warning: Work under a laboratory hood. Wear thick gloves, special
glasses, and a mask. Phenol can cause burns and is very toxic when
mixed.

a) From liquid phenol

Add 0.1% 8-hydroxyquinoline and 10% m-cresol to liquid phenol.


Shake 1 hour. To stabilize pH, wash 3 or 4 times with 1M Tris-HCl
buffer, pH 8.0; then with 0.1 M Tris-HCl pH 8.0 until the pH of the
solution is over 7.6 (add buffer, mix, wait until phases separate,
and discard the solution).

To prepare 1 M Tris-HCl pH 8.0, dissolve 121.1 g Tris in 800 ml


distilled water. Adjust pH to 8.0 with 0.1 N HCl (approximately 29
ml) and add distilled water to adjust final volume to 1,000 ml.
Autoclave.

b) From crystallized phenol (for 500 g)

Add 140 ml distilled water directly to the bottle containing phenol.


When dissolved, add 10% m-cresol and shake for 1 hour. Add
0.68 g 8-hydroxyquinoline and shake for 1 hour. Leave overnight at
room temperature.

Next day, adjust pH as for liquid phenol. Store at 4oC in amber


glass bottles. Under these conditions the saturated phenol is stable
for one or two months.

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Recommended Literature
Boulton, R.E., G.I. Jellis, D.C. Baulcombe and A.M. Squire. 1986. The
application of complementary DNA probes to routine virus detection
with particular reference to potato viruses. In: Developments and
applications in virus testing. R.A.C. Jones and L. Torrance. (eds.),
Great Britain, pp. 41–53.
Melton, D.A., P.A. Krieg, M.R. Rebagliati, T. Maniatis, K. Zinn, and M.R.
Green. 1984. Efficient in-vitro synthesis of biologically active RNA
and RNA hybridization probes from plasmids containing a
bacteriophage SP6 promoter. Nucleic Acids Research 12: 7035–
7056.
Owens, R.A. and T.O. Diener. 1981. Sensitive and rapid diagnosis of
potato spindle tuber viroid disease by nucleic acid hybridization.
Science 213: 670–672.

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