3.2.1 Total RNA Isolation
The isolation of total RNA was performed using acid guanidinium thiocyanate-phenol-chloroform extraction following the protocol of Chomczynski (Chomczynski and Sacchi, 1987).
During the process of RNA isolation from cells and tissues it is important to inactivate any RNAses. Therefore, all buffers were prepared with H2ODEPC and all materials consisting of glass or metal were baked for at least 8 h at 180°C.
For D.rerio RNA isolation 1 - 2 fishes were euthanized, the ovaries were removed as fast as possible on ice and transferred to a reaction tube with 500 µl ice cooled Sol D.
RNA from N.furzeri was isolated from whole fish. Therefore, the beheaded fishes were scaled off and finally pulverized in liquid nitrogen. Then the powder was transferred to a reaction tube containing 500 µl ice cold Sol D.
Alternatively, RNA of the Nothobranchius species and medaka were isolated from ovaries and tissues.
The following procedure was identical for all starting materials. All centrifugation steps were carried out at 4°C in a table-top centrifuge at maximum speed for 15 min, all working steps were performed on ice and all buffers and solutions were pre-chilled. Variations are mentioned.
The material in Sol D was carefully resuspended via needles with decreasing diameters. Following, 50 µl 2 M sodium acetate (pH 4), 500 µl phenol and 100 µl chloroform : isoamyl alcohol (24 : 1) were successive added and tilted over.
The reaction tube was stored for 15 min on ice and afterwards centrifuged for 20 min at 17300 xg at 4°C. The supernatant was carefully transferred to a new reaction tube and 500 µl ice cold isopropanol was added. Subsequently, the tube was stored for 1 h at -20°C and then centrifuged. The supernatant was removed and the pellet was resuspended in 200 µl Sol D. Following 400 µl isopropanol was added. The solution was stored for at least 1 h at -20°C and then centrifuged. After removing the supernatant the pellet was resuspended in 400 µl 70 % (v/v) ethanol and incubated for 15 min at room temperature in order to wash the RNA. After a final centrifugation the pellet was dried carefully with nitrogen and resuspended in 50 – 200 µl H2ODEPC.
3.2.2 Reverse Transcription
This technique was used to reverse-transcribe RNA in cDNA using SuperScriptIII Reverse Transcriptase. The newly synthesized cDNA can then be used as template for specific DNA amplification via PCR.
The reverse transcription was performed according the manufacturers´ protocol in 20 µl reaction mix. In a first step specific primers (2 pmol) or oligo(dT) (500 ng), 1 µl total RNA, 1 µl 10 mM dNTPs were mixed and added to 13 µl with
H2O. After 5 min at 65°C and 1 min on ice 4 µl 5x First-Strand Buffer, 1 µl 0.1 M DTT, 1 µl RNase – Inhibitor and 1 µl SuperScriptIII (200 u / µl) were added.
Subsequently, the mixture was incubated for 60 min at 40 – 50°C and then heat-inactivated for 15 min at 70°C.
3.2.3 Polymerase Chain Reaction (PCR)
PCR was used for amplification of specific cDNA produced by reverse transcription as well as for colony screening of transformed E.coli K12 DH5α.
The volume of the PCR was 10 µl or 50 µl depending on the purpose of the PCR: 10 µl PCRs were performed for colony screening and for testing varying parameters (e.g. annealing temperature, new primers, etc.). 50 µl PCRs were used to achieve higher amounts of PCR product as needed for sequencing or further processing.
For 10 µl or 50 µl PCR following components were applied:
Component Taq HS KOD HS
10x Buffer (B1 / KOD HS) 1.0 µl 5.0 µl 1.0 µl 5.0 µl
25 mM MgCl2 1.0 µl 5.0 µl - -
25 mM MgSO4 - - 0.6 µl 3.0 µl
2 mM dNTPs 1.0 µl 5.0 µl 1.0 µl 5.0 µl
10 µM forward Primer 0.7 µl 3.5 µl 0.3 µl 3.0 µl
10 µM reverse Primer 0.7 µl 3.5 µl 0.3 µl 3.0 µl
Template
(bacterial material / product of RT, dC-tailed cDNA)
Bacteria from agar plate /
0.5 µl 1.0 - 5.0 µl 0.5 - 1 µl 1.5 µl Polymerase (Taq, 5 u / µl /
KOD HS, 1 u / µl) 0.1 µl 0.5 µl 0.2 µl 1.0 µl
H2O ad 10.0 µl ad 50.0 µl ad 10.0 µl ad 50.0 µl
Table 3-1 Composition of Taq HS-PCR and KOD HS-PCR
The PCR procedure was carried out in a 96well Thermocycler (BioRad). For KOD HS polymerase the initial denaturation and polymerase activation occurred at 95°C for 2 min. During each cycle denaturation time was only 20 sec, followed by a annealing procedure for 10 sec at a temperature depending on the used primers (generally: Tm – 3°C). The extension was carried out at 70°C, the time was depending on the size of the desired DNA fragment and varied between 15 sec to 2 min. The amplification was repeated 25 – 30 times. The following summary table includes also reaction parameters for the Taq polymerase:
3.2.4 Rapid Amplification of cDNA Ends (RACE)
This technique is used to obtain full length sequence of an RNA transcript. It is used for amplification of nucleic acid sequence from mRNA between a defined internal site and either the 3´- or 5´- end (Frohman et al., 1988).
3.2.4.1 3´- RACE
The 3´-RACE uses the poly(A) tail of mRNA as priming site. For the reverse transcription (3.2.2) the Adapterprimer (33) was used binding to the poly(A) tail and additionally possessing a specific sequence. Following the RNA template was removed by adding 1 µl RNase H and incubation for 20 min at 37°C. The DNA amplification was carried out by PCR using a gene-specific internal forward primer and the Abridged Universal Amplification primer (AUAPrimer
(34)) which binds to the specific sequence of the Adapterprimer and a Taq polymerase (3.2.3). The PCR product was sequenced by MWG-Biotech and analyzed using GeneiousPro.
3.2.4.2 5´- RACE
5´-RACE was carried out using the 5´RACE System for Rapid Amplification of cDNA Ends following the manufacturer´s protocol with some adaptations:
For First-Strand cDNA synthesis GSP1 (gene-specific primer) (41) were used.
2.5 µl of GSP1 (1 µM), 2 µl RNA and 11 µl H2ODEPC were mixed. This mix was incubated for 10 min at 70°C and afterwards 1 min on ice. Then 2.5 µl 10x PCR buffer, 2.5 µl MgCl2 (25 mM), 1 µl dNTP mix (10 mM) and 2.5 µl DTT (0.1 M) were added, mixed and incubated for 1 min at 42°C. 1 µl SuperScriptIII RT was added and then incubated for 50 min at 42°C. Afterwards the reaction was inactivated for 15 min at 70°C. For the removal of RNA the reaction mix was heated up to 37°C and 1 µl RNase mix was added and incubated for 30 min.
The next step was the S.N.A.P. column purification of the cDNA. Then the purified sample was tailed with TdT (terminal deoxynucleotidyl transferase) and dCTPs. Therefore, 6.5 µl H2ODEPC, 5 µl 5x tailing buffer, 2.5 µl dCTP (2 mM) and 10 µl of the purified sample were mixed and incubated for 2.5 min at 94°C and 1 min on ice. Then 1 µl TdT was added and incubated for 10 min at 37°C and inactivated at 65°C for 10 min. The tailed cDNA was used in PCR for amplification (3.2.3). Therefore, Taq polymerase, the reverse primer (36) or (38) and as forward primer AAP (Abridged Anchor Primer) was used. One half of the PCR product was analyzed on an agarose gel the second part was diluted 1:20 and 1:100 with TE buffer and a second nested PCR was performed with UAP (Universal Amplification Primer) or AUAP (Abridged Universal Amplification Primer) as forward primer and (36) or (38) as reverse primer. Subsequently, the PCR product was sent for sequencing.
3.2.5 Determination of DNA / RNA Concentration and Purity
The nucleic acids concentrations were determined photometrically using NanoDrop 2000c (Thermo Scientific). Therefore, an absorption spectrum was recorded from 220 nm to 320 nm. The highest absorption of nucleic acids is at 260 nm. To calculate the concentration of DNA, the following formula was used:
260 ⋅ ⋅50
An indication of the purity of the DNA gives the equation:
8
An indication of the purity of the RNA gives the equation:
0 negatively charged DNA / RNA is separated via an electrical field, the migration velocity dependents on DNA size, structure, gel concentration and voltage.
Agarose was mixed with 1x TAE in concentration from 0.5 – 1.5 % and heated up until completely dissolved. The solution was poured on a gel tray and a comb for slot formation was appropriately inserted. After polymerization the gel tray was transferred to an electrophoresis chamber (BioRad) containing 1x TAE. The samples were mixed with 6x Loading Dye and loaded after removing the comb. Additionally, an appropriate marker was loaded in one of the slots.
The gel was run at 100 V until the bromophenol blue front had reached lower third of the gel. To visualize the DNA / RNA the gel was stained with ethidium
bromide, an intercalating fluorescent compound for 7 min. Afterwards, superfluous ethidium bromide was washed out twice for at least 5 min in water.
The analysis took place under a UV light (254 nm) and pictures were taken using ChemiDocTM MP Imager (BioRad).
For RNA size separation 1x TAE was prepared using H2ODEPC and agarose was used in a concentration of 1 %. Comb, gel tray and chamber were incubated with 10 % H2O2 for 30 min to destroy RNAses.
3.2.7 DNA Extraction from Agarose Gels
A possibility to isolate fragments of a defined size is to separate DNA by agarose gel electrophoresis, cut out the respective band and extract DNA from the agarose.
To avoid damage of the DNA by ethidium bromide and UV light the samples were loaded on two different slots on the agarose gel, a small amount to visualize the location of desired DNA and a larger amount for extraction. After running the gel, it was cut between the two lanes and only one half was stained with ethidium bromide. Under UV light the band was marked. Subsequently, the parts of the gel were aligned again and DNA in the unstained gel was cut out and purified with a Gel Extraction Kit (Qiagen) following the manufacturer´s protocol.
3.2.8 Dephosphorylation of DNA Ends
To avoid re-ligation of a vector DNA after restriction digest, the 5´-terminal phosphate was removed. This was achieved using Shrimp alkaline phosphatase (SAP).
The total volume was 10 µl containing 1 µl 10x SAP reaction buffer, 0.5 µl SAP, 5 µl linearized vector and H2O. After an hour at 37°C the enzyme was inactivated at 70°C for 20 min.
3.2.9 Ligation
This technique was used to clone a linear DNA fragment into a linearized vector. ATP-dependent T4 DNA Ligase is able to catalyze the ligation of blunt and sticky DNA ends. The reaction mix contained 1 µl 10x T4 DNA Ligase Buffer, insert and vector in a ratio of 3 : 1, 0.5 µl T4 DNA Ligase and water ad 10 µl. Insert and vector were mixed and heated for 5 min at 55°C to disrupt secondary structures of the DNA endings. Following the mix was stored on ice for 1 min and then the remaining components were added. The ligation mix was incubated at 4°C o/n and afterwards 1 h at room temperature. Subsequently the ligase was inactivated for 15 min at 65°C.
3.2.10 Restriction of DNA
Restriction enzymes are able to recognize and cut plasmid DNA and DNA fragments at defined base sequences. This can result in blunt ends or sticky ends with 5´- or 3´-overhang, depending on the specific restriction enzyme. This technique was used to open up plasmids at a defined position or as double or triple digest for DNA fragment isolation.
The reactions volume was 10 µl in general, consisting of 1 µl 10x CutSmart Buffer, 0.3 – 0.5 µl of the respective enzyme, 1 – 2 µl DNA (approximately 0.5 – 1 µg) and H2O (ad 10 µl). The reaction mixtures were incubated at least 1.5 h or o/n at 37°C, optionally inactivated and then analyzed by agarose gel electrophoresis.
3.2.11 Sequencing
To verify the correct insertion of DNA fragments into cloning vectors and to get information about the base sequence the resulting plasmid was sequenced. The sequencing was performed by GATC Biotech (Konstanz, Germany) or Microsynth AG (Balgach, Switzerland).
To sequence PCR products, samples were sent to Eurofins MWG Operon (Ebersberg, Germany) or Microsynth AG.