Dot Blot for Probe Quantification
DIG RNA Probe Dot Blot Quantification Procedure
1. Construct serial dilutions of the DIG labeled control RNA and both of your experimental RNA probes (T7 and SP6) as follows:
Tube 1: 1/5; Tube 2: 1/25; Tube 3: 1/125; Tube 4: 1/625; Tube 5: 1/3125
These dilutions work for me but you can do different dilutions if you like. Make the dilutions up in DEPC dH2O
2. Cut a piece of nitrocellulose membrane to be large enough to spot all of your samples, but small enough to easily fit inside the lid of a micropipette tip box (this will be your wash tray. Do not reuse as a tip lid).
3. Using a pencil, mark the membrane so that the locations of each of the dots will be clear. List the sample type at left and the dilution at the top. Note: the first dot will be 1 ul of undiluted sample, making six rows in total.
4. Spot 1 ul of each dilution for each sample so that they line up with both their sample types (rows) and dilutions (columns). Note, the samples will become invisible once dried.
5. Once dried, place the nitrocellulose membrane face down on a transilluminator and turn it on for 1 minute (do this with the door closed).
6. Incubate membrane in roughly 20 ml of blocking solution (MA B + 1.0% BSA) for 30 min. Use enough blocking solution to cover the membrane. Make sure to make enough blocking solution to dilute the anti DIG Ab in step 8.
7. Wash 3x 10 min each in PBT
8. Incubate with anti DIG antibody at 1:2000 in PBT for 1 hour.
9. Wash 3 x 10 min each in PBT
10. Incubate the membrane without agitation in the BCIP staining solution. Color may develop within minutes, or could take up to an hour.
11. Once development has proceeded the filter can be washed in PBT, dried, wrapped in plastic wrap and taped into notebook.
12. Determine the concentration of probe by comparing control dots of known mass to the dots made by experimental probes. For instance, given that the RNA probe control is provided at a concentration of 100 ng/ul, if the 1/125 control labeled RNA dot is the same intensity as the 1/25 T7 dot, we can infer that the concentration of the T7 solution is 20 ng /ul. I usually run my ISHes with probes at 0.5 - 1.0 ug/ml of HYB.
Notes: Conduct washes with membrane face up. Solutions can be poured off while the membrane remains adhered to the container by surface tension. PBT, MAB and BCIP staining solutions are the same as in the ISH protocol.
