Northern Blot Analysis CURRENT STATUS :


 The whole workflow for the quantification of specific transcripts by Northern blot analysis is described in detail, including RNA isolation, probe generation via labelling with Dig-dUTP, hybridization, signal visualization and quantification.


Introduction
For the analysis of (differential) gene expression or for the determination of translational efficiencies it is necessary to quantify the levels of specific transcripts.
A variety of methods are available, e.g. qRT-PCR(or RT-qPCR) and Northern blot analyses for single transcripts [1;2]. For highly parallel quantification of many transcripts RNA-Seq or DNA microarray analysis can be used. Northern blot analysis offers several important advantages in comparison to qRT-PCR, i.e. 1) the size of the transcript is determined and only non-degraded full-length transcripts are quantified, 2) it is clarified whether the gene of interest is transcribed into one specific transcript or whether several transcripts of different lengths exist (e.g. monocistronic and polycistronic), and 3) no expensive equipment is needed. First, the desired DNA sequence for probe generation is amplified via a "normal" PCR reaction. If necessary, the PCR fragment is purified. It is used as a template for the labelling reaction.

2.
The labeling takes place via a second PCR reaction with Dig-dUTP (1µl/ 100µl PCR reaction) and a LowT dNTP-mix to ensure efficient incorporation of the label.
The elongation time should be twice as long as for a "normal" PCR, because the incorporation of Dig-dUTP is slower than that of unlabeled nucleotides.
3. The incorporation of the label is verified by analytical gel electrophoresis, the probe must have a higher apparent molecular weight than an unlabeled negative 5 control.
Denaturing RNA gel electrophoresis 4. Prepare a denaturing RNA gel. The example is given for a gel of 8 cm x 12 cm with a volume of 80 ml. 0.8 g -1.6 g agarose are taken to yield concentrations of

5.
Put the hardened agarose gel into a horizontal gel electrophoresis system with 1x MOPS buffer as running buffer.

6.
Mix the RNA samples (2-5 µg) and the RiboRuler RNA size marker (1µl) with three volumes of RNA loading buffer. Heat the samples to 60°C for 10 minutes to denature RNAs, and subsequently put them on ice to inhibit refolding.

7.
Apply the samples to the gel and run for 2h at 100V.

8.
After the gel run, verify the integrity and the amounts of the ribosomal RNAs (23S, 16S). Document the gel picture, because the rRNA amounts of the different samples are used for normalization.
Blotting the RNA gel to a nylon membrane 9. The transfer of the RNAs from the gel to a nylon membrane occurs via downward capillary blotting. One or a few hours are sufficient, nevertheless, usually the blotting is performed overnight. Figure 1 gives an overview of the blotting 6 setup; the following points give a step by step description. 20. The membrane is stained with methylene blue to visualize the RNA size marker.
The positions of the size marker fragments are marked with a pencil. The membrane can be directly used for hybridization, or it can be dried and stored for later use.
Hybridization of the membrane 1.
Put the blotted membrane into a hybridization tube with 10 ml hybridization buffer and 120 µg/ml yeast tRNA, incubate for 60 minutes at 50°C in the hybridization oven with slow rotation.
2. 30 µl of Dig-labelled probe is incubated at 95 o C for 5 minutes to denature the probe, and then cooled on ice to inhibit renaturation. It is given to 10 ml hybridization buffer. The probe-free hybridization solution is removed from the hybridization tube and is discarded. The solution with the probe is added, and the tube is mounted again in the hybridization oven. It is incubated at 50 o C overnight with slow rotation.

3.
The hybridization solution with the probe is removed from the hybridization tube. It can be stored at -20°C and reused several times. Each time the probe has to be denatured before it is given to another membrane.

4.
The membrane is washed twice for 5 minutes at room temperature with 50 ml 2x SSC buffer + 0.1% (w/v) SDS at high rotation.

5.
The membrane is then washed twice for 15 minutes at 50°C with 1x SSC buffer + 0.5% (w/v) SDS again at high rotation speed.

6.
The membrane is then incubated once in freshly prepared wash buffer, and it is transferred to 20 ml blocking solution and incubated for 30 minutes at room temperature and slow rotation.
Quantifying the specific transcript level 7. 1 µl anti-Dig-AP antibody are given to 20 ml blocking solution. The mixture is incubated with the membrane for 30 minutes at room temperature under slow 8 rotation.
8. The membrane is washed three times for 10 minutes in 60 ml wash buffer at room temperature and fast rotation.

9.
The membrane is equilibrated in 20 ml detection buffer at room temperature for 5 minutes under slow rotation.  Translational coupling via termination-reinitiation in archaea and bacteria by Madeleine Huber, Guilhem Faure, Sebastian Laass, +6