COSTO BENEFICIO EN LECHONES DESPUÉS DEL USO DE LA

manifold consists of 3 blocks of perspex which are connected with 8 locating bolts. The top 2 blocks have a grid of 96 wells ( 8 x 1 2 ) which are 4 mm in diameter this allows the size of the dots to be controlled. The membrane is mounted between the top 2 blocks after prior equilibration in xlO SSC ( 66% w / v NaCl, 33% w / v tri- Na citrate, pH 7.0 ), dot blots require a high salt concentration to facilitate the effective tr ansfer of RNA from solution to membrane as this alters the physical state of the RNA making it 'sticky'. Once in close proximity to the membrane there will also be some charge interaction with the membrane which has a positive charge which increases binding of the RNA to the membrane.To facilitate unifonn suction and prevent leakage into adjacent wells a piece of Whatman 3 MM paper also equilibrated in x 10 SSC and was placed between the first and second perspex block under the membrane. RNA samples ( 500ng ) in a denaturing loading buffer were heated to 65°C for 15 minutes and rapidly cooled on ice. Ten volumes ( 400 ql ) of xlO SSC were added to the RNA samples which are loaded into the wells on the manifold and the vacuum applied. The wells were rinsed with two washes of xlO SSC ( 400ql ) to wash the filter and remove any RNA adhered to the side of the well wall. The membrane was removed from the manifold and treated as in section 2.11.

2.13. Nucleic acid hvbridisation

Nucleic acid hybridisation can be defined as two complementary single stranded polynucleotides, one target and one probe, combining in a sense - anti-sense fashion to form a stable duplex molecule under specific conditions.

As one of the least well understood areas of molecular biology this complex process has a number of factors affecting the efficiency and rate of duplex formation. These aie discussed in Chapter 4. The influence of each of these factors is dependent on the physical state of the nucleic acids involved. In this case the target nucleic acids are immobilized onto a solid support and then immersed in an aqueous solution containing the hybridisation components ( Northern, Dot blot analyses ).

Figure 2.2

Structural view of the BRL dot blot manifold: 1 .locating bolts, 2.sample well template ( 6mm diameter, 13mm Depth ), 3.Zeta-probe GT membrane, 4.filter support template,

5.Vacuum chamber.

N.B. Whatmann 3MM paper is inserted between 3. Zeta-probe GT membrane, 4.filter support template.

2.14. Hybridisation methods

The hybridisation method used comprised of 4 major stages ;

i.Pre-hybridisation ; pre-incubation of the membrane with a high concentration of calf thymus DNA and yeast total RNA to reduce the numbers of non-specific binding sites for the probe.

ii.Hybridisation ; incubation with a specific labelled DNA probe complementary to the target ribonucleic acid in essentially the same buffer as pre-hybridisation.

iii.Washing ; washing through a step-wise series of decreasing ionic stiength buffers which remove non-specifically bound probe.

iv.Autoradiography ; incubation with X-ray film which is then developed to obtain a photographic image of the blot giving the location and a semi-quantitative estimation of the amount of labelled probe bound to the specific nucleic acid target

on the membrane.

2.14.1. Pre-Hvbridisation

For pre-hybridisations the membrane was incubated in a solution compromising of 50% formamide v / v , IM NaCl, 0.05 volumesDenhardt's soln.( 0.1% Ficoll Type 400 w / v , 0.1% w /v polyvinylpyrrolidone, 0.1% w /v Bovine Semm Albumin Fraction V ), 1% w /v SDS, 50mM sodium phosphate pH 6.8 with HCl containing

0.5 mg / ml sonicated calf thymus DNA and 0.5 mg / ml sonicated yeast total RNA. The pre-hybridisation solution ( 20ml ) was pre-heated in a Techne HB-1 hybridiser ( Techne Ltd., Cambriodge, UK ) before addition into Techne hybridisation bottles ( boro-silicate ) containing the membrane ( 140mm X 100mm ). Both stock solutions of 10 mg / ml sonicated calf thymus DNA and 10 mg / ml sonicated yeast total RNA.were denatmed at 100°C for 5 minutes and cooled rapidly on ice prior to addition to the pre-hybridisation solution. The membrane was incubated for a minimum of 4 hours but usually overnight in pre-hybridisation solution. The hybridisation bottles rotated in the hybridiser at a rate of 4 revolutions per minute.

2.14.2. Hybridisation

The radiolabelled probe DNA ( see section 2.8.8 ) was denatured in boiling water for 5 minutes, cooled rapidly on ice and added to the pre-hybridisation solution. The pre-hybridised membrane was further incubated for 12-16 hours with the labelled DNA probe. Utilizing the same pre-hybridisation solution appeared to have no detrimental effects on the hybridisation.

2.14.3. Washing

After the hybridisation incubation the solution was removed and the membrane was washed at the hybridisation temperature in a series of NaCl / SDS buffers. The buffers comprised of a series of decreasing NaCl concentrations ;

Wash 1 : 44.5mM NaCl, 0.5% SDS Wash 2 : 44.5mM NaCl, 0.1% SDS Wash 3 : 23.9mM NaCl, 0.1% SDS Wash 4 : 12.8mM NaCl, 0.1% SDS

Membranes were washed for 1 hour with 30 ml of Wash 1 followed by 45 minutes in each of the consecutive Washes 2-4 using the same volume. The degree of removal of non-specific background was estimated using a gieger counter ( mini-1,

series 900 ) and the membrane was washed until a satisfactory level of background was achieved ( 1-5 radioactive counts per second ).

2.15. Autoradiography

After washing, membranes were removed from the hybridisation bottles and sealed whilst wet into plastic bags. The sealed membranes were placed into Mult-e-role autoradiography cassettes and secured using masking tape. The cassettes contained Hi-Speed-X intensifying screens to improve signal intensity. The X-ray film ( Hyperfilm-MP ) and screens used for autoradiography were from Amersham International Ltd, Chalfont, UK. All manipulations involving the film were carried out in a dark room to avoid any pre-exposure of the film. A corner of the film was cut

off to aid in identification of the orientation of the fihii and the film placed in the cassette with the membrane. Cassettes were incubated at -70°C as this improves the intensity of signal 5 fold ( Young et al, 1984 ). The radioactivity passing thi'ough the X-ray film hits the intensifying screen which emits photons of light that are captured by the silver halide in the X-ray film ( Maniatis et al, 1982 ). Füms were incubated for different lengths of time dependent on the signal that had been deteraiined from the geiger counter. In general, signals of greater than 10 radioactive counts per second ( cps ) were incubated for 16-24 hours, 5-10 cps for 48 hours and less than 5 cps for 72-96 hours. After incubation the films were developed in Kodak LX 24 developer ( Kodak Ltd., Slough, UK ) for 5 minutes, fixed in Kodak Unifix for 5 minutes and then washed under running tap water. The films were dried and analysed.

2.16. Analysis / Quantification of Autoradiographic signals

The autoradiographic images on the X-ray film were analysed using a flying spot scanning densitometer ( Shimadzu model CS-9000, Shimadzu Corp., Kyoto,

Japan ). The scanner moves the developed X-ray film over a light beam and measures the absorption of light at a pre-deteimined wavelength, in this case a wavelength of 550nm was used. For dot blots the absorption was calculated and the area under the peak of absorbance was calculated. The units of area are equal to 1 unit of area = an absorbance of 0.001 OD units eveiy 0.02mm. The amount of radioactivity contained on the image was quantified against a set of radioactive standard dot blots which were incubated at the same time as the experimental membrane so the area obtained using the densitometer was expressed as counts per minute ( cpm ) per dot.