User Manual

Section 6

RNA Blotting

RNA must be denatured prior to application to Zeta-Probe

or nitrocellulose membranes to

ensure optimal hybridization. Two protocols are presented for denaturing RNA samples.

6.1 Alkaline RNA Denaturation and Fixation

1. Always wear gloves when handling blotting membranes. Pre-wet the blotting membrane

by placing it gently at a 45° angle into a tray of wetting solution. The Zeta-Probe membrane

is wetted in distilled water, nitrocellulose is wetted in 6x SSC (see Section 9 for solution

preparation).

2. Assemble the Bio-Dot apparatus according to the instructions in Section 3.1. Remember

to apply the vacuum and then retighten the screws that hold the apparatus together.

3. Immediately before use, dissolve RNA samples in 500 µl of ice-cold 10 mM NaOH, 1 mM

EDTA.

4. Samples and wash solutions may be applied with a standard pipet or a Costar

Octapette pipet. Apply the denatured RNA, and pull the sample through by passive

filtration or by applying a gentle vacuum.

Note: A method for applying gentle vacuum to the apparatus is to adjust the flow valve

to setting 3. Use a finger to cover the valve port exposed to air. The amount of vacuum

reaching the manifold will be regulated by the pressure of your finger on the valve.

5. Rinse all wells to wash through any sample on the side of the wells. Rinse with 500 µl

cold 10 mM NaOH, 1 mM EDTA. Apply vacuum (flow valve setting 1, Figure 3) until the

sample wells are dry.

6. Disassemble the Bio-Dot apparatus. Remove the blotted membrane and rinse it

in 2x SSC, 0.1% sodium dodecyl sulfate (SDS). Nitrocellulose membranes must

be baked under vacuum for 2 hours at 80° before hybridization. The Zeta-Probe

membrane is ready for hybridization. If hybridization is not to be undertaken

within 2 days, then bake the Zeta-Probe membrane under vacuum for 30 minutes

at 80°C. The Zeta-Probe membrane and nitrocellulose membranes can be stored

dry between two pieces of filter paper in plastic bags at 23–25°C.

6.2 Glyoxal RNA Denaturation and Fixation

1. Prepare RNA samples to the following final concentrations:

50% dimethyl sulfoxide (DMSO)

10 mM NaH

, pH 7.0

1 M glyoxal

2. Incubate the RNA for 1 hour at 50°C. Cool the samples on ice.

3. Always wear gloves when handling blotting membranes. Prewet the blotting membrane

by placing it gently at a 45° angle into a tray of wetting solution. The Zeta-Probe

membrane is wetted in distilled water, nitrocellulose is wetted in 6x SSC (see Section

10 for solution preparation).