Electrophoresis of RNA — Agarose gel containing formaldehyde

1. Day 2 Day 3 Day 1 Electrophoresis of RNA—Agarose gel containing formaldehyde Preparation of RNA samples from K562 cells Agarose gel analysis for RT-PCR product RNA samples (provided): human blood cells K562 SW48 Reverse transcription Polymerase chain reaction Vacuum Transfer to Nylon membrane Positive control (provided) & negative control Wash Pre-hybridization Detection UV Crosslinking Hybridization (probe provided)

2. Day 1 Prepare the RNA samples Samples: human blood cells RNA 5 ug/5ml K562 RNA 10 ug/5ml SW48 RNA 10 ug/5ml Mix 3 volumes freshly made Loading buffer with 1 volume of each RNA sample Denature the RNA/Loading buffer mixtures at 65oC, 10 min Immediately chill the denatured samples on ice for 1 min Add 2 ml Loading dye Load the samples on the gel Prepare the Agarose/formaldehyde gel 0.6 g Agarose 3.784 ml 10x MOPS buffer 34 ml H2O Melt the gel by microwave Add 2.16 ml Formaldehyde (37%) in fume hood Run the gel (in hood) in 1x MOPS buffer at 70V for about 2 hr Stain the gel in EtBr UV examination Show the migration distance with a ruler beside the gel. 18S rRNs range in 1.8 kb to 2.0 kb 28S rRNs range in 4.6 kb to 5.3 kb

3. Vacuum transfer of RNA onto a Nylon membrane Pour on solution 3, Neutralising solution. Leave for 7 min. Soak the gel twice (2 x 15 min) in 20xSSC (Solution 4) Pour on Solution 4, Transfer solution. Leave for 1 hr. Pretreated the nylon membrane by wetting with water and then floating in 20x SSC for 20 min. Place the membrane on a filter paper. Assembly the Vacuum blotting unit UV crosslinking Allow the membrane to dry, wrapped in filter papers and aluminum foil loosely, stored at room temperature. Pour solution 2, Denaturation solution, onto the center of the gel. Use enough solution to cover the gel. Leave for 7 min. Ensure that the gel remains covered with solution during the treatment. During the denaturation step, stablise the vacuum at 50 mbar.

4. (600 ul) Day 2 (AM) Isolation of total RNA from K562 cells 所有離心都以最高轉速1分鐘 Add b-ME before use. Use 600 ml to disrupt the cells, Dilute 50 x OD260/OD280 detection

5. Reverse transcription (RT) 5x cDNA synthesis buffer 4 ml 0.1M DTT 1 ml RNaseOUT (40 U/ml) 1 ml DEPC-treated water 1 ml 10 mM dNTP mix 2 ml SuperScript III (15 units/ml) 1 ml Primer (50 mM oligo(dT)20) 1ml RNA (from K562, 10 pg-5 mg) x ml DEPC-treated water to 10 ml 65oC, 5 min, then chilled on ice, Spin Final 20 ml 50oC 50 min (50-60oC, 30-60 min) 85oC 5min (RNase H digestion 37oC 20 min)

6. Polymerase chain reaction (PCR) 95oC 72oC 4oC 2min 7min  94oC 70oC 72oC 10sec 30sec 1min H2O 5.25 10X Buffer 1.25 25mM Mg solu 1 2.5 mM dNTP 1 Taq DNA polymerase 1 (Promega, 0.25U/ml) Primer F (HglbFa 5 mM) 1 Primer R (HglbRb 5mM) 1 Template 1 2 mM Mg 35 cycles 0.2 mM dNTP Day 3 0.4 mM primer Run 1.5% Agarose Gel 12.5 ml Positive control: K562 cDNA Negative control: EtBr stain, UV examine

7. Day 2 (PM) Pre-hybridization and hybridization for the Northern blot Pre-heat 15 ml DIG Easy Hyb at 50oC Incubate the blot with gentle agitation for 30 min at 50oC in 8 ml of pre-heat DIG Easy Hyb Denature DIG-labeled DNA probe (provided) by boiling for 10 min and rapidly cooling on ice-water.Add to 5ml of pre-heat DIG Easy Hyb and mix well but avoid foaming. Pour of prehybridization solution and immediately add probe/DIG Easy Hyb mixture to membrane Incubate the blot with gentle agitation at 50oC overnight

8. Day 3 Immunological detection with CSPD Wash Wash the blot in cold wash buffer at RT for 2 x 5 min (30ml) (10ml) (30ml) Wash the blot in hot wash buffer at 68oC for 2 x 15 min (8ml) (1ml) cold wash buffer: 2x SSC, 0.1% SDS hot wash buffer: 0.1x SSC, 0.1% SDS

9. 組別 姓名 學號 姓名 學號 1. Please attach the figure of the gel electrophoresis separation for the RNA samples. (Please indicate the 28S and 18S RNAs) 2. The total quantity of the RNA sample prepared from K562 cells:___________, the concentration:_____________, and the OD260/OD280 ration:______.

10. 3. Please attach the figure of gel electrophoresis analysis for the RT-PCR products. (Please label the bp numers of the size standards) 4. Please attach the figure of the Northern blotting result. Predict the sizes of the positive signals.