Making RNA probes for in situ hybridization
Make DNA templates via PCR
Day 1
It's preferable to start with constructs that contain RNA polymerase start sites (T3, T7, or SP6) which allow use of primers outside the start-point. This gives the enzyme some docking space, improving transcription efficiency. If this is not possible, use extended primers with RNA-polymerase start sites in their 5' tails.
Purify PCR products with QiaQuick PCR purification kit and resuspend in nuclease free water.Make DNP-NTP mixture (if needed)
Day 1
| Amount | Final concentration | |
| Sigma H20 | 5.7 μl | |
| ATP 100mM | 2 μl | 10 mM |
| CTP 100mM | 2 μl | 10 mM |
| GTP 100mM | 2 μl | 10 mM |
| UTP 100mM | 1.3 μl | 6.5 mM |
| DNP-11-UTP 10mM | 7.0 μl | 3.5 mM |
In vitro transcription reaction
Day 1
| 10 μl reaction | 20 μl reaction | |
| Sigma H20 to final vol | ||
| 10X Buffer | 1 μl | 2 μl |
| 10X NTP mix | 1 μl | 2 μl |
| RNAse inhibitor | 0.7 μl | 1.3 μl |
| template DNA | >150ng | >300ng |
| RNA polymerase | 1 μl | 2 μl |
Heat 10X Buffer to dissolve salts before using.
Incubate at 37oC for 2-4 hours.
Eliminate template
Day 1
Add 1 μl DNAseI (RNAse free) to each reaction.
Incubate for 15 min at 37oC. This is a time sensitive step.
Precipitate probes
Day 1 (immediately after eliminating the template)
Bring reaction to 50μl with RNAse free water.
Add 1/10th volume 3M NaOAc pH5.2 (5μl)
Add 2.5 volumes of 100% EtOH (125μl)
Store at -20oC for at least 2 hours.
Spin 20 min at 4oC at 13200 rpm.
Remove supernatant.
Pellet for DIG/biotin reactions will be white, DNP reactions will be yellow and FITC reactions will be orange.
Add 250-400μl 70% EtOH and spin 20 min at 4oC at 13200 rpm.
Remove supernatant and repeat wash with 100% EtOH.
For a 20μl reaction with a visible pellet, it is usually sufficient to dissolve the pellet directly in 100μl 50% formamide.
If necessary: resuspend probes in 50 μl of RNAse free water. Take 5 μl for quantitation. Then add 45 μl
100% formamide and store at -20oC.
For 10μl reaction : with visible pellet, it is sufficient to dissolve them directly in 50μl 50% formamide in RNAse free water.
Properly dissolved probes that have sufficiently high concentrations typically foam a lot. It will take a couple of minutes to dissolve a probe.
Making RNA probes for in situ hybridization page 2of 2
