RNA techniques

5.4.1 Isolation of RNA

Total-RNA is isolated according to the RNeasy protocol (QIAGEN). The culture medium is removed and the cells washed twice with PBS. 600µl RTL buffer supplemented with 6µl ß- mercaptoethanol are added. The cells are removed with a rubber policeman and transferred into a microfuge tube. DNA is destroyed by homogenizing the lysates 5-8 times through a G20 needle. After mixing with 600 µl of 70% ethanol, the lysates are transferred onto RNeasy microspin columns and centrifuged for 15s at 10,000 rpm (Microcentrifuge MiniSpin, Eppendorf). The flow-through is discarded and the columns washed with 700 µl of RW1 buffer by centrifugation. The collection tube is replaced and the columns are treated twice with 500 µl RPE buffer and centrifuged. The flow-through is discarded and the columns centrifuged at 14,000 rpm for 2 min to remove all residual buffer. The total RNA is eluted with 50µl RNase free water and collected in Rnase-free microfuge tubes.

5.4.2 Separation of dsRNA

RNA-molecules are separated via gel electrophoresis under RNase free conditions on 1% - 1.5% agarose in TAE with 40µl/l ethidium bromide of gel for detection (see 5.3.2).

5.4.3 RT-PCR

To obtain cDNAs from mRNA or to estimate the levels of mRNA in cells, RNA transcripts can be converted into copy-DNA (cDNA) by reverse transcriptase. In subsequent PCR steps, the cDNA strands are amplified to yield detectable levels. Prior to RT-PCR, RNA templates are heated to 75°C for 5 min to denature the RNAs, and cooled on ice to prevent re-annealing. RT-PCRs are carried out with the Titan-Kit (Roche) according to the manufacturer´s protocol.

Mastermix1: 1 µl dNTPs (10 mM) 1 µl primer1 (25 µM) 1 µl primer2 (25 µM) 2.5 µl DTT (100 mM) 0.5 µl RNasin (40u/µl) 10 µl template RNA (100 µg/ml) 14 µl RNase free water

Mastermix2: 10 µl RT-PCR buffer (5x) 5 µl RNase free water 0.8 µl enzyme-mix

The two mastermixes are combined in a 500µl tube, placed in a thermocycler (PTC-200, Biozym Diagnostics), and incubated according to the following program:

reverse transcription: 50°C 30:00 min denaturation: 94°C 00:30 min

annealing: 62°C 00:45 min 30 cycles elongation: 68°C 01:00 min

last extension: 68°C 10:00 min

5.4.4 In vitro generation of 5´-thiol-modified siRNAs with T7-polymerase

The antisense strand is generated according to the RiboMAX-protocol (Promega). To generate the 5‘-thiol-modified sense strand an 8fold excess of GSMP over the non-modified NTPs is added to the reaction mixture. 1-2µl of RNasin are added to compensate for the non- RNase-free synthesis procedures. GSMP is synthesized as described in chapters 3.1.1 and 3.1.2. The purified product is dissolved in 10 mM Tris-HCl, pH 7.5. Following the in vitro transcription reaction, DNase is added to both reaction mixtures to degrade the DNA templates. Also, 4 µl of CIP are added to remove 5´-triphosphates and to release the thiol- function from the 5‘-thiophosphate group of the sense strand. After 15 min of incubation at 37°C, the 21mers are precipitated and dried according to the manufacturer´s manual. The RNA pellet is dissolved in Rnase-free water, and the concentration determined photometrically. Equimolar amounts of sense and antisense strand are pipetted together, supplemented with the corresponding amount of 10x annealing buffer, heated to 75°C for 5 min, and allowed to cool to RT over several hours for hybridization.

5.4.5 Reduction of homodimers of 5´-thiol-modified siRNAs

Under oxidizing conditions, free thiol groups may react to form disulfide bonds that link two monomers together. To enable the coupling of siRNA and CPPs, these spontaneously formed homodimers have to be reduced.

Synthetic siRNA aliquots of 20 nmol are dissolved in 120 µl of RNase free 0.1 M DTT solution (Titan-Kit, Roche). Enzymatically generated siRNAs are supplemented with ¼ of the total volume of 100 mM DTT-solution (Roche). Both reaction mixtures are incubated at 37°C for at least one hour.

To remove the DTT, the reduced siRNAs are purified by gel filtration (MicroSpinTM G-25 Columns, AP-Biotech) according to the manufacturer’s manual.

5.4.6 Coupling of synthetic siRNA and CPP

After over night incubation with DTT, synthetic thiol-modified siRNAs are purified by gel filtration via MicrospinTM G-25 colums (AP-Biotech). In the last step of the procedure, the siRNA aliquot is eluted into an equimolar amount of the activated peptide. The reaction mixture is incubated 1 h at 37°C.

5.4.7 Coupling of enzymatically synthesized siRNA and CPP

After gel filtration of the enzymatically synthesized siRNA, the concentration is determined to avoid errors due to free nucleotides. An equimolar amount of peptide solution is diluted in EB to the same volume and both solutions are pipetted together under argon. Precipitate forming at high concentrations due to aggregation of pepsiRNAs can be dissolved by the addition of NaCl-solution to a final concentration of up to 400 mM.

5.4.8 Radiolabelling of siRNAs

T4-kinase attaches the terminal phosphate residue of [32P]-γ-ATP to free 5´-hydroxyl-groups of oligonucleotides. The lyophilized enzyme is dissolved in 25 µl of water. After 5 min of quelling, 50-100 pmol of siRNA and water are added to yield 24 µl. After the addition of 1 µl of [32P]-γ-ATP (10 mCi/µl) the reaction mixture is incubated at 37°C for 30 min and the reaction stopped with 5 µl of 250 mM EDTA. Residual ATP is removed by gel filtration (MicroSpinTM G-25 Columns, AP-Biotech) according to the manufacturer´s manual.

5.4.9 Analysis of siRNAs on sequencing gels

siRNAs are radioactively labeled and run on a 25 cm x 55 cm 10% TBE-urea sequencing gel of 0.1 mm thickness. The Macromould glass plate is prepared with 3 x 200µl of binding- silane and the thermoplate is coated with 1 ml of repel silane. Both plates are thoroughly cleaned with ethanol to remove an excess of silane.

The 12% gel is prepared by dissolving 42g of urea in 10 ml of 10x TBE and 25 ml of water by gentle heating. After cooling down, 30 ml of 40% acrylamide solution is added and the solution filtered to remove undissolved solid. For polymerization and cross-linking, 0.8 ml of APS solution and 40 µl of TEMED are added. Prior to running, the gel is heated to 50°C via the thermoplate of the gel chamber, and 3000V/28mA are applied for 30 min to equilibrate the gel with the TBE buffer.

The 10µl samples are prepared by adding 10µl of formamide loading buffer, heating to 95°C for 5 min. The samples are placed on ice. 3-6 µl of sample are loaded and the gel is run at 3000 V at 21-27 mA. The gel is fixed by soaking in 10% acetic acid for 30 min, washed with water for 5 min, and dried at 70°C for 45-60 min. The detection of the radioactivity is performed using a phosphorimager screen (BAS III, Fuji). Radioactivity screens are read out on a Bas reader (Fujix Bas 1000, Fuji) and the results evaluated on Tina 2.0 software.

0.5M NaOH is used to remove the gel and binding-silane from the carrier-glass plate.