Molecular biology

2.3.1 Concentration determination of nucleic acids

The concentration determination of nucleic acids is based on the absorption maximum of the aromatic rings present in the nucleic acids at a wavelength of 260 nm. To determine the concentration of nucleic acids, its absorption extinction in an aqueous solution was measured at wavelengths 260 nm and 280 nm in comparison to the corresponding buffer without nucleic acids using a NanoDrop spectrophotometer according to the manufacturer’s instructions. The concentration was then calculated using pre-determined absorption values at 260 nm, as described (Sambrook et al., 1989). The ratio of OD₂₆₀/OD₂₈₀ determined the purity of the nucleic acid solution, which is usually 2.0 for pure RNA or oligonucleotides, and 1.8 for pure DNA.

Lower ratios indicate the presence of protein, phenol, or other impurities, which must be removed by PCI-extraction (2.2.2).

1 OD260 = 50 µg / ml double-stranded DNA 1 OD260 = 33 µg / ml single-stranded DNA 1 OD260 = 40 µg / ml single-stranded RNA

2.3.2 Agarose gel electrophoresis of nucleic acids

To analyse DNA probes against different snRNAs, agarose gel electrophoresis of nucleic acids was performed as described (Sambrook et al., 1989). Depending on the length of the oligos, our gels contained 1 % (w/v) agarose and 0.4 µg/ml ethidiumbromide in 1x TBE buffer. Samples were supplemented with a 6x DNA loading dye. The nucleic acids were separated in a horizontal gel running assembly at 100V in 1x TBE buffer. The bands were visualized with UV-light at 254 nm.

Migration rates of the marker dyes through agarose gels

% agarose (w/v) xylene cyanol bromophenol blue

0.7-1-7 4000 bp 300 bp

2.5-3.0 800 bp 100 bp

2.3.3 Proteinase K digestion

To obtain a protein-free RNA sample, proteinase K digestion was performed. To each sample reaction, 2 µl of the stock solution (proteinase K mix) was added. The proteinase K digestion contained final concentration as - 0.2% (w/v) SDS, 10 mM EDTA, and 0.3 mg/ml proteinase K.

All sample reactions were incubated for 30 min at 37°C. RNA was extracted by PCI-extraction and precipitated.

2.3.4 Denaturing polyacrylamide gel electrophoresis of RNA

To separate different snRNAs present in the tri-snRNP fractions, denaturing polyacrylamide gel-electrophoresis was used. The gels contained 8 M urea, 0.5x TBE, and according to the size of the snRNA, the concentration of polyacrylamide was kept 8%. The gels were polymerized with 50 µl 10% (w/v) APS and 5 µl TEMED per 10 ml gel solution. RNA samples were resuspended in 10 µl of RNA loading buffer and denatured for 5 min at 95°C and immediately transferred on ice. The electrophoresis was carried out in 1x TBE buffer at 500V until the xylene cyanol reached the bottom of the gel. The RNA fragments on the gel were visualized by silver staining or by SYBR^TM Gold nucleic acid staining method.

Migration rates of marker dyes in denaturing polyacrylamide gels polyacrylamide (%) xylene cyanol bromophenol blue

5.0 130 bases 35 bases

6.0 106 bases 29 bases

8.0 76 bases 26 bases

10.0 55 bases 12 bases

20.0 28 bases 8 bases

2.3.5 Silver staining of RNA gels

Silver staining of RNA gels was performed according to Merril et al. (Merril et al., 1981). During the procedure, at least a 10-times the gel volume was used for all solutions. Sample containing gel was fixed in a solution of 40% methanol and 10% acetic acid for at least 30 min. After fixating,

the gel was washed twice with a solution containing 10% ethanol, 5% acetic acid for 15 min to eliminate interfering substances. Then, the gel was washed with ddH₂O once and subsequently stained in 12 mM AgNO₃ solution for 30 min. After incubation with the staining solution, the gel was washed twice with ddH₂O twice. Then, the gel was quickly treated with the developing solution (0.28 M Na₂CO₃, 0.0185% formaldehyde) to remove excess silver from the surface.

Carefully discard the solution and again added a fresh developing solution. Developing was stopped by adding a 5% acetic acid solution. The gel was transferred to a Whatman paper and dried under vacuum at 80°C for 1 hr.

2.3.6 SYBR^TM Gold nucleic acid staining

SYBR^TM Gold staining of RNA gels was performed according to manufacturer’s protocol after fixation of in 40% (v/v) Methanol / 10% (v/v) acetic acid. After staining the gel, RNA was visualized with Fujifilm FLA-7000 laser scanner at a wavelength of 495 nm.

2.3.7 Radioactively labelled DNA-probes synthesis for Northern analysis

To precisely detect tri-snRNP U snRNAs by the Northern blot analysis, radioactively labelled DNA probes were produced from their corresponding DNA templates with the help of the

‘Prime It II Random Primer Labeling Kit’ (Stratagene). DNA templates were (kindly provided by Dr. Patrizia Fabrizio from our department at MPI-BPC) analysed for degradation by an agarose gel. For DNA probes synthesis, 25 - 50 ng of DNA template was mixed with 23 µl ddH₂O and 10 µl of random ninemer primers and were denatured at 95°C for 5 min. Subsequently, the mix was cooled down at room temperature, followed by short centrifugation in order to allow hybridization. The reaction mixture was then supplied with 10 µl of dATP primer buffer, 5 µl of [α-³²P] dATP (3000 Ci/mmol), and five units of Klenow enzyme. Then, the reaction mixture was incubated for 10 min at 40°C. After completion of the required time period, 2 µl of 0.5 mM EDTA solution was added to stop the reaction. The DNA-probes were then isolated from free radioactive dATP by using a ‘ProbeQuant^TM G-50’ columns as per the manufacturers' directions.

For Northern blot analysis, typically 10-20x 10⁶ cpm of the labelled DNA-probes were used.

Alternatively, oligonucleotides were 5' end-labelled with [gamma-³²P] ATP (6000Ci/mmol) using the T4 PNK (Polynucleotide Kinase) from NEB, as per the manufacturer’s instructions.

2.3.8 Northern blot analysis

Northern blot analysis was carried out to detect a specific snRNA in the tri-snRNP sample. For this, samples were first separated by denaturing polyacrylamide gel electrophoresis (as described in section 2.2.2.4) and transferred onto a Biodyne nylon membrane (Pall Corp.) using a semi-dry transferring method (PeqLab Biotechnologie GmbH). For semi-dry transfer, 2 Whatman paper sheets soaked in 0.5 x TBE buffer were placed on the cathode plate, followed by the pre-wet nylon membrane, gel, and again 2 more pre-soaked Whatman filter paper sheets. The sequence of forming the transfer sandwich was carefully followed precisely in the described order. After gently removing the bubbles from the transfer setup, finally, the anode plate was softly placed onto the transfer sandwich. Gradually screws were tightened, and the transfer unit was connected with the power supply. The transfer was carried out for 2 hr at room temperature at a constant current supply of 2 mA/cm². After completion of the RNA transfer, the unit was carefully opened to retrieve the nylon membrane. With the RNA side facing up, the membrane was exposed to UV irradiation using the Stratalinker 2400 (from Stratagene, USA) to crosslink the RNA with the membrane.

Radiolabelled DNA probes were generated using the protocol described in section 2.2.2.7. The RNA containing membrane was pre-hybridized for at least 2hr at 37°C in the hybridization buffer containing denatured fish sperm DNA. After that, denatured radiolabelled probes were added, and the incubation was continued overnight at 42°C. The next morning, the membrane was washed two times with 25 ml of 2xSSC, and 0.5% SDS solution for 1 min. And twice again, with 25 ml 2xSSC, and 0.1% SDS solution. All washings were done at room temperature. In the final step, the washed membrane was dried and wrapped in the transparent plastic foil. The membrane was then visualized by exposing onto the phosphorimaging screen or the X-ray film for sufficient duration.

2.4 Cell culture methods