REQUERIMIENTOS DE DISEÑO

3.2.3.1 RNA extraction

The abdomen from 3 days old female flies was cut off and the remaining somatic portion comprising head and thorax was ground while the ovaries were hand-dissected and also isolated by grinding the tissue in Trizol using a pistil (Sigma Aldrich; Taufkirchen, Germany). RNA was extracted with Trizol (Invitrogen; Carlsbad/CA, USA) according to the manufacturer’s instructions and quantified using spectrophotometry.

3.2.3.2 Beta (β)-elimination of RNA

40 µg total RNA dissolved in 40.5 µl H2O was incubated with 12 µl 5x borate buffer (148 mM borax, 148 mM

boric acid pH 8.6) and 7.5 µl NaIO4 (200mM dissolved in H2O) for 10 min at RT. The oxidation was quenched

by addition of 6 µl 100% glycerol (10 min, RT). The elimination was performed by elevating the pH with 2M NaOH (5-7 µl) (ensure that pH=12). After 90 min at 45°C the sample was transferred to a Mini quick spin

oligo column (Roche Diagnostics; Mannheim, Germany), and centrifuged (12 000 g, 2 min). 20-40 µg glycogen were added and RNA was precipitated with 3x volume 100% ethanol (12 000 g, 15 min). RNA pellet was washed three times with 70% ethanol (last step 4°C, o/n) and dissolved in 20 µl 2x denaturating gel loading buffer. The samples were analyzed on a 15% Sequagel Acrylamide-Urea gel and subsequently used for generation of Solexa sequencing libraries.

3.2.3.3 Northern blotting

1-5 µg of RNA were separated on a 20% Sequagel Acrylamid-Urea Gel (National Diagnostics; Atlanta/USA) at 250 V for 90 min. RNA transfer was performed on a positively charged Nylon membrane (Roche Diagnostics; Mannheim, Germany) by semi dry blotting for 30 minutes at 20 V. Crosslinking of the RNA to the membrane was achieved by irradiation with UV-light. Membranes were transferred into hybridization tubes and pre-hybridized in Church buffer (1% (w/v) bovine serum albumine, 1 mM EDTA, 0.5 M phosphate buffer, 7% (w/v) SDS, pH 7.2) for at least 2 hours at 37°C in an oven under constant rotation. The probes were labeled by incubating 9 μl H2O, 2 μl 10x PNK buffer, 2 μl 5 mM probe oligonucleotide (=10 pmol), 1 μl

PNK (Fermentas) and 6 μl [γ-32P] ATP for 1h at 37°C. Unbound radioactive nucleotides were removed using a Sephadex G-25 spin column (Roche Diagnostics; Mannheim, Germany). Hybridization with labeled as- DNA-probes was performed overnight at 37°C in 5 ml Church buffer. Membranes were washed three times for 20 minutes with 2xSSC buffer with 0,1 % SDS and exposed on Phosphoimager Screens (FujiFilm; Tokio, Japan) for up to 1 week. Screens were scanned using a Typhoon scanner (Amersham Biosciences) and band intensities were analyzed using Multi Gauge software (Fujifilm; Tokyo, Japan).

Stripping of the membrane was achieved by dipping it into boiling 1% SDS solution by incubating it for 5 minutes in the solution. After a second pre-hybridization the membrane was reused for hybridization with further probes.

3.2.3.4 Analysis of miRNA and mRNA by quantitative RT-PCR (qPCR) 3.2.3.4.1 miRNA Profiling

The microRNA content of synchronized cells in various cell cycle phases (G1, late S and G2) was analyzed with qRT-PCR on an ABI Prism 7000 sequence detection system (Applied Biosystems Life Technologies, Carlsbad / CA, USA). Reverse transcription was performed using the miScript system (Qiagen, Hilden, Germany) according to the miScript protocol.

Reaction mix for reverse transcription:

miScript RT buffer (5x) 4 μl

100 ng RNA 0.3 – 0.7 μl

miScript enzyme mix 1 μl

H2O 14.3 – 14.7 μl

Samples were incubated at 37°C for 60 min and then inactivated at 95°C for 5 min. 100 μl of water were added to make a final volume of 120 μl.

The qPCR reaction mixes for 14 reactions (for 1 row of 96-well plate): SyBr-Green Mastermix (2x) 70 μl

miScript universal primer (5 µM) 14 μl miScript specific primers (10 µM) 7 μl

H2O 35 μl

9 μl of reaction mix and 1 μl of RT-reaction per well was amplified in an ABI PRISM 7000 qPCR cycler (Applied Biosystems; Foster City, USA) using the following conditions:

15 min 94°C initial denaturation ---

40 cycles:

20 sec, 94°C denaturation 30 sec, 55°C annealing 30 sec, 70°C extension

The primer sequences miRNA amplification can be found in chapter 3.1.10.4. Cycle of threshold values (CT- values) were usually determined via the auto-CT function and manually adjusted if necessary. Expression was quantified with the 2-(ΔΔCt) method (Livak and Schmittgen 2001).

3.2.3.4.2 mRNA levels

3.2.3.4.2.1 Digestion of DNA

Endonucleolytic digestion of DNA was carried out with endonuclease DNase I acquired from Thermo Fisher Scientific (Waltham, USA) and the buffer from Fermentas (St. Leon-Rot, Germany).

RNA 5 μg

DNase I 1 μl

DNase buffer (10x) 5 μl

RiboLock 1 μl

H2O add 50 μl

After incubation at 37°C for 30 min, 1 μl Proteinase K was added and incubated for 15 min at 65°C in shaking incubator at 600 rpm. The reaction mix was supplemented with equal volume of Phenol/Chloroform/IAA, pH 4.5-5 (Roth; Karlsruhe, Germany), vortexed and centrifuged (full speed, 20 min). The supernatant was precipitated with 80 μl isopropanol and 1 μl glycogen and incubated at RT for 10 min. The reaction mix was centrifuged at 12 000 g for 10 min. The pellet was washed with 100 μl 70% ethanol, dried at 55-60°C for 10 min and resuspended in100 µl RNase free water.

3.2.3.4.2.2 mRNA profiling

100 ng of total RNA after digestion of DNA was reverse transcribed according to the Superscript II Reverse Transcriptase protocol (Invitrogen; Karlsruhe, Germany) primed with random primer (Eurofins MWG Operon).

random primer (100 µM) 1.58 μl

100 ng RNA x μl

dNTP Mix (10 mM each) 1 μl

H2O add 12 μl

The mixture was heated to 65°C for 5 min and quick chilled on ice. The contents of the tube were briefly centrifuged. Then following components were added:

First-Strand Buffer (5x) 4 μl

DTT (0.1 M) 2 μl

RiboLock RNase inhibitor 1 μl

SuperScript II RT 1 μl

The contents of the tube were mixed gently and incubated at 42°C for 50 min. The reaction was inactivated by heating at 70°C for 15 min. 100 μl of water were added to get a final volume of 120 μl. The qPCR reaction mix was as follows, according to the DyNAmo Flash SYBR Green qPCR Kit (Thermo Fisher Scientific; Waltham, USA).

Reaction mix for one well of a 96-well plate:

Dynamo Flash Master Mix 5 µl

oligo_s (10µM) 0.5 µl

oligo_as (10µM) 0.5 µl

H2O 2.9 µl

xylencyanolblue (0.03%) 0.1 µl

9µl of the reaction solution was aliquoted in each well of a 96 well plate using an 8-canal pipette. 1 µl of the template was added and the samples cycled on a TOptical Thermocycler (Biometra; Jena, Germany) using the following PCR-program:

10 sec, 50°C

3 min 95°C initial denaturation ---

40 cycles:

30 sec, 95°C denaturation 30 sec, 59°C annealing 42 sec, 72°C extension

The primer sequences mRNA amplification can be found in chapter 3.1.11.8. Cycle of Threshold values (CT- values) were usually determined via the auto-CT function. Expression was quantified with the 2-(ΔΔCt)

method (Livak and Schmittgen 2001).