negative control either if the corresponding gene is not
expressed in the analyzed tissue or if the probe is transcribed
from a scrambled sequence.
4. Alternatively, the SP6 RNA polymerase can be used for
probe synthesis. In this case the SP6 promoter sequence
(5
0 -ATTTAGGTGACACTATAG-3
0 ) must be added to the
reverse primer. Sometimes, the transcription efficiency by SP6
polymerase is higher than by T7 polymerase.
5. To generate a sense probe, based on gene orientation, the
promoter sequence is added to the forward primer.
6. Alternatively, to the template generation by PCR, the amplified
mRNA region can be cloned into a plasmid, which contains T7
and SP6 RNA polymerase promoter sequences in the flanking
regions. To use the plasmid DNA as template for the following
in vitro transcription, it needs to be linearized at unique recognition sites for restriction enzymes creating blunt end or
5
0 -overhangs. Those restriction sites should be flanking the
insert and located as close to the inserted sequence as possible.
No terminator region flanks the insert, so the in vitro transcription stops when the template end is reached. The sequence
between insert and cutting site will therefore be transcribed
as well.
7. For double-labeling experiments a fluorescein- or biotinlabeled probe is needed.
8. Store probe in aliquots at À80
C and avoid repeated freeze/
thaw cycles.
9. Longer probes (>500 nts) can have negative effects on hybridization efficiencies or tissue penetration depth. The size can
be reduced by hydrolysis to generate an optimal probe length
of ~300–500 nts. For that propose, add 1 volume of freshly
prepared alkaline hydrolysis buffer and incubate at 60
C for a
calculated time (t) ((t [min] ¼ (probe length [kb]Àdesired
probe length [kb])/(0.11 Â probe length [kb] Â desired
probe length [kb])) [5]. Add acetic acid to a final concentration
of 0.5% (v/v) and sodium acetate to a final concentration of
0.1 M to stop hydrolysis. Add 1 volume of isopropanol and
incubate for !30 min at À80
C. Centrifuge (!16,000 rcf) for
30 min at 4
C. Wash pellet with 80% (v/v) ethanol. Centrifuge
(!16,000 rcf) for 10 min at 4
C. Dissolve pellet after drying in
100 μL RNase-free double-distilled water.
10. To make a denaturing formaldehyde gel in MOPS buffer, melt
1 g of agarose in 72 mL of deionized water, add 10 mL of 10Â
MOPS buffer and mix, add 18 mL of 37% formaldehyde in a
fume hood and mix thoroughly, and pour the gel. Mix RNA
samples and RNA ladder with RNA-loading dye, denaturate
the samples at 70
C for 10 min, and chill on ice for 3 min
18
Thomas Dresselhaus and Andrea Bleckmann
expressed in the analyzed tissue or if the probe is transcribed
from a scrambled sequence.
4. Alternatively, the SP6 RNA polymerase can be used for
probe synthesis. In this case the SP6 promoter sequence
(5
0 -ATTTAGGTGACACTATAG-3
0 ) must be added to the
reverse primer. Sometimes, the transcription efficiency by SP6
polymerase is higher than by T7 polymerase.
5. To generate a sense probe, based on gene orientation, the
promoter sequence is added to the forward primer.
6. Alternatively, to the template generation by PCR, the amplified
mRNA region can be cloned into a plasmid, which contains T7
and SP6 RNA polymerase promoter sequences in the flanking
regions. To use the plasmid DNA as template for the following
in vitro transcription, it needs to be linearized at unique recognition sites for restriction enzymes creating blunt end or
5
0 -overhangs. Those restriction sites should be flanking the
insert and located as close to the inserted sequence as possible.
No terminator region flanks the insert, so the in vitro transcription stops when the template end is reached. The sequence
between insert and cutting site will therefore be transcribed
as well.
7. For double-labeling experiments a fluorescein- or biotinlabeled probe is needed.
8. Store probe in aliquots at À80
C and avoid repeated freeze/
thaw cycles.
9. Longer probes (>500 nts) can have negative effects on hybridization efficiencies or tissue penetration depth. The size can
be reduced by hydrolysis to generate an optimal probe length
of ~300–500 nts. For that propose, add 1 volume of freshly
prepared alkaline hydrolysis buffer and incubate at 60
C for a
calculated time (t) ((t [min] ¼ (probe length [kb]Àdesired
probe length [kb])/(0.11 Â probe length [kb] Â desired
probe length [kb])) [5]. Add acetic acid to a final concentration
of 0.5% (v/v) and sodium acetate to a final concentration of
0.1 M to stop hydrolysis. Add 1 volume of isopropanol and
incubate for !30 min at À80
C. Centrifuge (!16,000 rcf) for
30 min at 4
C. Wash pellet with 80% (v/v) ethanol. Centrifuge
(!16,000 rcf) for 10 min at 4
C. Dissolve pellet after drying in
100 μL RNase-free double-distilled water.
10. To make a denaturing formaldehyde gel in MOPS buffer, melt
1 g of agarose in 72 mL of deionized water, add 10 mL of 10Â
MOPS buffer and mix, add 18 mL of 37% formaldehyde in a
fume hood and mix thoroughly, and pour the gel. Mix RNA
samples and RNA ladder with RNA-loading dye, denaturate
the samples at 70
C for 10 min, and chill on ice for 3 min
18
Thomas Dresselhaus and Andrea Bleckmann