Bio-Rad Bio-Dot® and Bio-Dot SF Microfiltration Apparatus - Manual (Page 13)

Section 6
RNA Slot 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. 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 9 for solution
preparation).
2. Assemble the Bio-Dot SF 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 an 8-channel 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 three. 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 any sample on the side of the wells through. 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°C 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
2
PO
4
, pH 7.0
1 M glyoxal
2. Incubate the RNA for 1 hour at 50°C. Cool the samples on ice.
3. Pre-wet the blotting membrane by placing it gently at a 45° angle into a tray of wetting solution.
Zeta-Probe membrane is wetted in distilled water, nitrocellulose is wetted in 6x SSC (see
Section 9 for solution preparation). Always wear gloves when handling blotting membranes.
4. Assemble the Bio-Dot SF apparatus according to the instructions in Section 4.1. Remember
to apply the vacuum and then retighten the screws that hold the apparatus together.
5. Samples and wash solutions may be applied with a standard pipet or an 8-channel pipet.
Apply the denatured RNA, and pull the sample through by passive filtration or by applying a
gentle vacuum.
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