Analysis of RNA

S1 nuclease analysis was initially described as a method for the determination of transcription initiation sites (Berk and Sharp, 1977). Total cellular RNA or mRNA is hybridised to a C-³² P-ATP 5' end-labelled DNA probe that overlaps the transcriptional start site. The DNA-RNA hybrids are then digested with S1 nuclease (from Aspergillus oryzae), which digests single stranded nucleic acids. Thus, only the regions of homology where the RNA forms a stable double strand with the DNA probe are protected from nuclease digestion. The nucleic acids are then precipitated, denatured and separated on a polyacrylamide gel. The size of the DNA probe detected after digestion corresponds to location of the transcription initiation site.

For S1 analysis, 40 Hg of total RNA were ethanol precipitated to obtain an equal concentration of RNA for all samples. The RNA was resuspended in 10 Hl of hybridisation buffer (80%

formamide, 0.4 M NaCl, 40 mM Pipes pH 6.4, 5 mM EDTA). Subsequently, 1 Hl of aC-³²P-ATP end-labelled DNA probe was added. If the site of transcriptional initiation is to be determined, the DNA probe must be labelled at the 5' terminus, since a radioactive label at the 3' end would be lost from the DNA during S1 digestion.

mRNA 5'---3' Labelled DNA 3'---5'*

S1 nuclease digestion 9

mRNA 5'---3' Labelled DNA 3'---5'*

The hybridisation mix was incubated at 70°C for 30 minutes to resolve secondary structures in the RNA. The mix was then immediately transferred to a water bath set to 50°C and incubated overnight. The homologous DNA and RNA molecules hybridise at a temperature between 45°C and 50°C depending on the length of the region of homology between the nucleic acids. The samples were then chilled on ice and 100 Hl of a S1 digestion mix [containing S1-buffer (1.25 M NaCl; 0.15 M NaAc, pH 4.5; 50 mM ZnSO₄), 10 Hg/Hl small denaturated salmon sperm carrier DNA and 60 U nuclease S1] was added. The samples were then incubated at 30°C for 1

hour. The samples were cleaned by extracting once with phenol/chloroform and once with chloroform. The nucleic acids were than precipitated by adding 1 Hl tRNA (1 mg/ml), 250 Hl of ice cold ethanol (96%) and incubated for 1 hour at -80°C. The precipitated mixture was centrifuged (750 x g, 10 minutes at 4°C). The supernatent was discarted, 1 ml 70% ethanol added and centrifuged once again (750 x g, 10 minutes at 4°C). This step was repeated once more. The supernatant was again discarded and the pellet dried in vacuum dryer. The pellet was resuspended in 16 Hl sample buffer (USB). After denaturing the samples in a heating block at 95°C for 5 minutes, the nucleic acids were separated on a 6% denaturing polyacrylamide gel (Fig. 2.2). The gels were run at between 2000 volts and 2500 volts at a temperature of 50°C depending on the expected size of the labelled fragments. At the end of the electrophoresis the plates were taken out the apparatus dismantled and the gel attached to a Whatman paper. It was then dried on a gel vacuum dryer for 120 minutes at 80°C and exposed to phosphor imager plates (Fuji).

Figure 2.2: S1 Gel Apparatus

PCR Fragment length (kb) Elongation time

<1 1.5 3 4.5 6

45 secs 1 min 2 mins 3 mins 4 mins 2.2.5.2.2 Reverse Transcription-PCR (RT-PCR, Titan^TM One Tube RT-PCR System)

PCR was used as a sensitive technique to quantify the level of marker gene expression. RT-PCR was performed using a one step technique. In a one step RT-RT-PCR, both the cDNA synthesis reaction as well as the PCR reaction are performed with an optimised buffer (5 x, 7.5 mM MgCl_2, DMSO) and enzyme (AMV RT and Expand^TM High Fidelity enzyme mix) without requiring the addition of reagents between cDNA synthesis and the PCR. The one step reaction system uses the AMV RT for first strand cDNA synthesis and the Expand^TM High Fidelity enzyme blend (which consist of Taq DNA polymerase and Pwo DNA polymerase) for the PCR part. This technique allows amplification of fragments up to 2 kb with decreased error rates.

0.5 Hg of DNase treated RNA was used as a template and the primer concentration was 20 HM.

1Hl of enzyme mix, 1 Hl RNase inhibitor (40 U/Hl), 2.5 Hl DTT-solution (100 mM), 10 Hl 5 x RT-PCR buffer and 0.2 mM dNTPs were added per PCR reaction. The PCR reactions were performed with the following thermal cycling program [in a Gene Amp PCR system 9600 or 2400 (Perkin Elmer)]: incubation step for 30 minutes at 50°C, denature template at 94°C for 2 minutes; 10 x cycles: denaturation step: 30 seconds at 94°C; annealing: for 30 seconds (primer depending temperature); elongation: 45 seconds-4 minutes (depending on the size of the expected fragment) at 68°C (Tab. 2.1). The annealing temperatures are usually between 50-60°C and were calculated with computer software (Primer designer, version 2.0-3.0 from Scientific &

Educational Software). 25 x cycles (depending on the abundance of the respective mRNA):

denaturating step for 30 seconds at 94°C; annealing: 30 seconds (primer depending temperature);

elongation: 48 seconds-4 minutes (depending on the size of the expected fragment) at 68°C, and one single cycle of prolonged elongation (up to 7 minutes) at 68°C.

Table 2.1: RT- PCR Elongation Times 2.2.5.2.3 Real Time RT-PCR

Real Time RT-PCR was used to accurately quantify expression of hGH in WAP3hGH transgenic mice.

An accurate quantification using normal RT-PCR is not possible as here the amplification efficiency cannot be measured after each cycle, which would allow the samples to be compared with each other during the duration of the PCR amplification step. However, in the real time RT-PCR this is possible. The principle can be seen in figure 2.3.

Figure 2.3: Principle of Real Time PCR

In addition to the two primers normally present in the RT-PCR reaction, a probe that is labelled with two different fluorescent marker and that is specific to a sequence in between the binding sites of the two primers is also added to the reaction. When both markers are close to each other (i.e. the probe is intact) and the reaction is excited with an argon laser energy is transferred from the reporter (R) to the quencher (Q) which then emits light. However, during the PCR reaction both primers are extended through the action of Taq polymerase until they meet the probe. The probe is then displaced from the DNA and, via the 5' nuclease activity of the Taq, degraded. This means that the two markers are no longer next to each other and the energy transfer from the reporter to the quencher can no longer take place. Now both fluorescent markers can emit light.

The strength of the emission from the markers is proportional to the amount of DNA that has been amplified in each reaction. The fluorescence is measured once every 7 seconds allowing the PCR reaction to be accurately followed and, after comparison with a known standard, the amount of RNA/DNA in the sample to be calculated. As the quantification is made during the PCR run, there is no need for the samples to be loaded on to a gel and this, in turn, excludes a

source of sample contamination from the experiment.

The following ingredients were pipetted together for each reaction: 45 Hl master mix (contains reverse transcriptase and Taq polymerase, RT/PCR buffer, nucleotides, Mg²⁺, the 2 primers and the fluorescently marked probe) and 5 Hl of an RNA sample (or water as a negative control). The following PCR program was then run: reverse transcription for 45 mins at 48°C, inactivation of the RT activity and denaturing of the template for 2 mins at 94°C, 40 PCR cycles (15 secs 94°C, 1 min 60°C) then hold at 25°C.

2.2.5.2.4 Northern Blot (Lehrach et al., 1977)

Northern blot analysis is used to determine the size of transcription products. 20 Hg of total RNA was mixed with a 2 x loading buffer (Sigma), denatured for 15 minutes at 65°C and loaded on to a 1% formaldehyde agarose gel. RNA was then transferred immediately to a nylon membrane (Zeta-Probe, Bio-Rad) by capillary elution (Fig. 2.4).

Figure 2.4: Northern Blot Apparatus

One long piece of Whatman 3 MM paper was soaked in 20 x SSC and laid on a glass plate to form a support. Any air bubbles between the glass plate and the Whatman 3 MM paper were smoothed out with a glass rod. Whilst the gel was soaking in 10 x SSC (for 20 minutes at room temperature), the nylon membrane was washed in water_DEPC and then soaked in 10 x SSC. The gel was placed in an inverted position on the Whatman 3 MM paper. The nylon membrane was cut exactly to the size of the gel and was placed on top of the gel. Any air bubbles between gel and nylon membrane were smoothed out again. Three more pieces of Whatman 3 MM paper were cut out to the size of the gel and soaked in 20 x SSC before being placed on top of the nylon membrane. Finally, a stack of paper towels was mounted on top of the Whatman 3 MM papers. A glass plate was placed on the top and weighted down with a 500 g weight. The capillary transfer was left to proceed overnight in the presence of 20 x SSC. After the transfer the filter was washed once for 10 minutes in 6 x SSC and cross-linked. The gel was stained in ethidium bromide to evaluate the efficiency of the RNA transfer. The membrane was then prehybridised for 4 hours at 42°C and subsequently hybridised overnight at 42°C.