33. TSA staining solution: Always prepare fresh following the
manufacturer’s instructions (e.g., TSA-Plus-Cyanine3-Kit;
PerkinElmer).
34. Fast Red staining solution: Always prepare fresh following the
manufacturer’s instructions (e.g., SIGMAFAST™ Fast Red
TR/Naphthol AS-MX tablets).
35. 100 mM Glycine pH 2, 0.1% (v/v) Tween
® 20.
36. Clearing solution: 70% (w/v) Chloral hydrate in 10% (v/v)
glycerol.
37. DAPI-containing anti-fading mounting media.
38. 77% (v/v) Glycerol in PBS.
3 Methods
3.1 RNase
Decontamination
of Solutions, Glassand Plasticware
1. Use sterile, disposable plasticware whenever possible—these
should be RNase free.
2. Decontaminate other plasticware from RNases by soaking in
3% (v/v) hydrogen peroxide for 10 min followed by extensively
rinsing with RNase-free double-distilled water.
3. Decontaminate glassware by baking at !180
C for several
hours (caution: use bottles with heat-resistant lids and
pour ring).
4. Treat all solutions that are not containing compounds with
primary amine groups with DEPC. Add 1 mL DEPC to 1 L
solution, stir for !2 h, and autoclave to inactivate DEPC.
3.2 In Vitro
Transcription
of Labeled Probes
1. Select a 300–500-nucleotide (nt)-long region on the mRNA,
which shows only low identity to related sequences (see Notes
2 and 3).
2. Design forward and reverse primers to amplify the DNA of
the selected region by PCR from reverse-transcribed mRNA.
To generate an antisense probe (complementary to the
in vivo-transcribed RNA), the reverse primer starts with
the
T7
RNA
polymerase
promoter
sequence
(5
0 -TAATACGACTCACTATAG-3
0 ) (see Notes 4–6).
3. Preform PCR reaction according to the manufacturer’s
instructions.
4. Clean up the PCR product by a DNA purification kit according
to the manufacturer’s instructions and elute in RNase-free
double-distilled water.
5. Set up a DIG RNA-labeling reaction according to the manufacturer’s instructions (e.g.: DIG RNA Labeling Kit (SP6/T7),
MERCK) (see Note 7).
10
Thomas Dresselhaus and Andrea Bleckmann