Dot Blotting RNA Probes

 

Materials (see ISH protocol for stock solution recipes):

·       cRNA Probes

·       DEPC-treated H₂O

·       RNA Standard dilution series (1:10 - 1:1000)

·       Positive-charge nylon membrane

·       0.1 M Phosphate buffer (PB, 1:2 0.2 M PB)

·       Blocking solution                                            for 1 ml

10 % normal sheep serum                   100 µL

2 mg/ml Bovine Serum Albumin         20 µl 100 mg/ml stock

0.3% Triton X-100                              100 µl 3% stock

in 0.1 M PB                                         500 µl 0.2 M PB

                                                            280 µl dH₂O

·       Anti-digoxigenin-AP or anti-fluorescein-AP Fab fragments

·       0.1 M Tris, pH 9.5 (1:10 1M Tris)

·       Coloration buffer                                             for 100 mL

0.1 M Tris, pH 9.5                                    10 ml 1M Tris, pH 9.5

50 mM MgCl₂                                1.01 g MgCL₂•6H₂O

100 mM NaCl                               2 ml 5 M NaCl

0.1% Tween-20                            1 ml 10% Tween-20

                                                      dH₂O to 100 ml

·       BCIP & NBT Stock Solutions (Boehringer)

 

Procedure:

 

1. Prepare serial ten-fold dilutions of RNA probes with DEPC H₂O to yield solutions of 1:10, 1:100 and 1:1000of the probe.Use 2ul of probe into 18 ul of DEPC water and then make serial dilutions.

 

1:1 1:10 1:100 1:1K 1:10K Shh           Ptc           Std

2. Cut out a small piece of membrane on which to apply the diluted probe and mark headings for placing dilutions with a pencil or sharpie.  Include a row/column for application of undiluted probe.  Be careful not to touch the membrane with your fingers.  An example (not to scale) might be:

 

 

 

 

3. Apply 1 µl of each probe and standard dilution to the proper region of the membrane.

 

4. Place membrane in properly-sized dish and then into 60° C oven until dry (wet dots will disappear.)

 

5. Wet membrane with 0.1 M PB.

 

6. Replace PB with blocking solution and incubate 10 minutes at room temp with gentle shaking.  A 60 mm dish will need 3-5 ml of solution to cover the membrane completely

 

7. Add Fab fragments to the blocking solution to make a 1:1000 dilution of antibody and continue to shake gently 20-30 minutes.

 

8. Wash membrane 2 x 5 min with 0.1 M PB and 1 x 5 min. with 0.1 M Tris, pH 7.4.

 

9. Wash membrane 2 x 5 min with coloration buffer.

 

10. Prepare an appropriate amount of coloration reaction buffer by adding 3.5 µl BCIP and 4.5 µl NBT to each ml of solution needed.  Incubate membrane in this solution 15-30 minutes in the dark until no more color seems to be developing on dots.

 

11. Rinse membrane with deionized water.

 

12. Estimate RNA probe concentrations by comparing the concentrations of the last visible dots of the standard and sample.  Fully concentrated RNA standard is 100 µg/ml.  Thus, if the last visible standard dot is 1:1000 and the last visible sample dot is 1:100, the sample is 10-fold less concetrated than the standard, or 10 µg/ml.  For ISH, probe is used at a concentration of 0.2-0.5 µg/ml, so this probe would be used at a 1:50 dilution in hybridization buffer.