Introduction
Northern blot analysis allows the detection and quantification of specific RNA species from a particular cell type. Isolated RNA is electrophoresed through an agarose/formaldehyde gel which separates the RNA species by size. The faster migrating RNA fragments are the smallest,however, the distance of migration is not linear, rather it is inversely proportional to the size RNA molecule. When RNA has separated following electrophoresis, it is stained with ethidium bromide and visualised using ultra violet light. For gels of total RNA the 28S and 18S ribosomal subunits are visible and act as convenient size markers (approx 4.8 and 1.9kb, respectively).
To probe for a specific mRNA species by northern blot, it is first necessary to transfer the RNA from the agarose/formaldehyde gel to a nylon membrane. RNA is detected by hybridisation using a labelled probe. The probe is a DNA or RNA molecule which is chemically or radioactively labelled. We will use a [³²P]-dUTP-labelled probe.

Timetable
1^st Day Prepare gel and electrophorese  Transfer o/n
2^nd Day Synthesize and purify probe    Pre-hybridize membrane & Hybridize with probe o/n
3^rd Day Wash membranes and expose to film o/n
4^th Day Develop film Ð interpret data

1^st Day (AM) Ð prepare and load gel
Materials required
RNase free water (i.e. DEPC treated)              Agarose
10 x running buffer (0.2 M MOPS pH 7.0,     Formaldehyde
10 mM EDTA)                                                UV transilluminator
Formamide                                                        Ethidium bromide
0.5 M iodoacetamide                                         Microfuge

Preparation of the samples
1. Keeping all samples on ice, add 10-20 µg of RNA from the control cells and/or treated cells to a sterile Eppendorf tube. The volume of RNA should be increased to 15 µl by the addition of DEPC treated water.
2. To this add 2.5 µl deionised formamide, 2.5 µl formaldehyde and 2.5 µl of running buffer(10 x stock), and 2.5 µl loading buffer
3. Denature samples by heating at 60ûC for 20 min, then snap cool on ice.

Gel Preparation
1. You will be provided with 130 ml molten agarose at 60 ûC (1.5 g agarose/130 ml H20).
2. Carefully add:      15.0 ml 10 x running buffer
                                 3.0 ml 0.5 M iodoacetamide
                                 2.25 ml 40% formaldehyde
                                 10.0 µl Ethidium bromide (10 mg/ml stock)
3. Mix by swirling and pour into sealed gel tray (don’t forget the comb to form the wells).

Load and run the gel
1. Load samples into the wells and run at 100 volts for 2 h (e.g. over lunch

1^st Day (PM) Ð stop gel and transfer o/n
Materials required
Hybond N (Nylon) membrane                  20 x SSC (0.3M Na3citrate, 3M NaCl)
Saran wrap (DOW chemical company)     Glass plate
Whatmann 3MM paper                            Bottle of water as weight for blot
Sponge soaked in 20 x SSC

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