Protocol ****

Denaturation

1. Prepare the following:

25 µg total RNA/ 1µg poly A⁺ RNA x µl

1 µg oligo dT₁₉V (2 µg/µl) 0.5 µl

8 µg oligo dT₃₀ (8µg/µl) 1.0 µl

GFP poly A⁺ RNA (10ng/µl) 0.5 µl

ddH₂O x µl

Final volume 10.5 µl

2. Heat at 70 °C for 10 minutes, cool to 42 °C, slowly.

Note: Heat in 70 °C heat block then take out block and allow to cool on bench top until temp is 42 °C.

Reverse Transcription

3. To sample from above add the following:

dNTP mix (800 µM dATP/dGTP/dTTP, 5 µM dCTP) 1.0 µl

5x First Strand Buffer 5.0 µl

10x DTT 2.5 µl

[α-³²P]-dCTP (10µCi/µl) 5.0 µl

SuperScript II (200 U/µl) 1.0 µl

Final volume 25.0 µl

4. Incubate at 42 °C for 1 hour.

Hydrolysis of RNA

5. Sequentially add the following:

1% SDS 1.0 µl

500 mM EDTA 1.0 µl

2 N NaOH 3.0 µl

6. Heat at 65 °C for 5 minutes

7. Then add the following:

1 M Tris.HCl pH 7.6 10.0 µl

2 N HCl 3.0 µl

8. Incubate at RT for 10 minutes.

9. Add ddH₂O 7.0 µl

Final volume 50.0 µl

10. Purify probe over G-50 Micro Column (Pre-spin col umn at 3500 rpm for 1 minute, apply probe to column, spin at 3500 rpm for 2 minutes.)

Note: Save 1µl for counting and 1µl for denaturing gel.

Prehyb membrane

11. Incubate filter at 65 °C for 6-20 hrs in 10 ml Hybridization solution (5x SSC, 5x Denhardt�s, 0.5% SDS, 100 µg/ml ssDNA)

5ml 10ml 15ml 20ml 25ml 30ml 50ml

20x SSC 1.25 2.50 3.75 5.00 6.25 7.50 12.5

50x Denhardt�s 0.50 1.00 1.50 2.00 2.50 3.00 5.00

20% SDS 0.13 0.25 0.38 0.50 0.63 0.75 1.25

ssDNA 0.05 0.10 0.15 0.20 0.25 0.30 0.50

ddH₂O 3.10 6.20 9.20 12.3 15.4 18.5 30.8

Probe preparation

12. Boil probe 5 minutes, place on ice at least 5 minutes.

13. Add 2 µg poly A₇₂ to 2 ml Hybridization solution.

14. Add denatured probe to the 2 ml Hybridization solution.

15. Incubate 1 hr at 65 °C.

16. Add 3 ml Hybridization solution.

Hyb membrane

17. Incubate filter in 5 ml Hybridization solution for 20 hrs at 65 °C.

Washes

18. Wash membrane 5 minutes at RT in 2x SSC/0.1% SDS.

19. Wash membrane 20 minutes at 65 °C in 2x SSC/0.1% SDS.

20. Wash membrane 1 hour at 65 °C in 0.1x SSC/0.1% SDS.

21 (Optional). If needed, repeat 1 hour at 65 °C 0.1x SSC/0.1% SDS.

Adapted from Huntsman Cancer Institute Katze Lab Protocols