From: Dr. David Wilkinson
Division of Developmental Neurobiology,
National Institute for Medical Research,
The Ridgeway,
Mill Hill, London NW7 lAA, UK.

A.Preparation of labelled RNA probe

1.Mix these reagents in the following order at room temperature:

Sterile distilled water	8.5 ul
5x transcription buffer	4.0 ul
0.1M dithiothreitol	2.0 ul
Nucleotide mix: (10mM wrt G,A,CTP and 6.5mM UTP, 3.5mM DIG-UTP.)	2.0 ul
Linearised plasmid (1 ug/ul)	1.5 ul
Placental ribonuclease inhibitor (100 U/ul)	0.5 ul
SP6, T7 or T3 RNA polymerase (10 U/ul)	1.5 ul

2. Incubate at 37 oC for 2 hr.
3. Remove a 1 u1 aliquot and run on an agarose gel contalning 0.5 ug/ml ethidium bromide to estimate the amount synthesized. This gel must be ribonuclease-free, but does not need to be denaturing. An RNA band 10-fold more intense than the plasmid band should be seen, indicating that 15 ug probe has been synthesized.
4. Add 2 u1 ribonuclease-free DNase 1(1 U/ul), and incubate at 37oC for 15 min. This step is optional.
5. If the transcript is greater than 1 kb in length, reduce the average size to 500 bases. Add an equal volume of 80 mM NaHCO3, 120 mM Na2CO3 and heat at 60oC for a period of time given by: time (min) = (L-0.5)/(0.055L), where L is the starting length of the transcript. It is advisable to check on an agarose gel the size of the product compared with a 500 bases transcript since over-degraded probes give low signals and high backgrounds.
6. Add 130 u1 dH2O, 50 ul 10 M ammonium acetate, 400 ul ethanol, mix and incubate at -20 oC for 30 min. Do not incubate at a lower temperature as this may precipitate unincorporated nucleotides.
7. Spin in a microfuge for 10 min, wash pellet with 70% ethanol and air-dry the pellet.
8. Redissolve in 150 u1 ice-cold dH2O, then add 50 ul 10 M ammonium acetate, 400 ul ethanol, incubate at -20 oC for 10-20 min, and repeat step 7.
9.Redissolve in ice-cold TE at -0.1 ug/ul and store at -70 oC. Use 1-5 ul for each ml of hybridisation mix.

* It better to use long probes as they are much more sensitive than short probes. Probes up to 3 kb work well, but it is beneficial to size reduce probes longer than 1 kb by partial hydrolysis. In general, we have found that even if the probe includes conserved coding sequences, moderate to high stringency conditions will eliminate cross-hybridisation to related genes.

Radioactive probes are essentially the same except that the only UTP source comes from the radio isotope and this is a very low concentration which, depending upon the specific activity may be inhibiting the polymerase. Look at the promega technical guide about IVT.

Purification may also be a little different as well. If you would like a slightly cleaner probe, then run it through a sephadex G50 column instead of double precipitation. We usually do this with the RA probes.