2. Materials and Methods
2.14 Polymerase-Chain-reaction (PCR)
The polymerase chain reaction is a standard method to amplify specific DNA fragments (Saiki et al. 1985, Mullis and Faloona 1987). Thereby double stranded DNA is denatured at high temperatures, which allows short oligonucleotides (primers) to bind to the single stranded DNA (annealing). Once bound, these oligonucleotides will be extended by the polymerase (elongation). The periodic repetition of the denaturation, annealing and elongation steps results in an exponential increase in DNA fragments. Whether the PCR was successful can be checked by agarose gel electrophoresis.
2.14.1 Standard PCR
The standard PCR was used to control the efficiency of the cDNA synthesis after reverse transcription PCR (Section 2.14.5). Therefore, specific primers for the housekeeping genes glyceraldehyde 3-phosphate dehydrogenase (GAPDH, for human samples) or hypoxanthine-guanine phosphoribosyltransferase (Hprt, for mouse samples) were used. The following reaction was used:
1 µl cDNA
5 µl 5x buffer
2.5 µl dNTPs (2 mM)
0.5 µl Primer fw (10 pmol/µl) 0.5 µl Primer rev (10 pmol/µl)
0.75 µl MgCl2 (50 mM)
0.7 µl Mango Taq DNA-Polymerase (1 U/µl) 14.05 µl ddH2O
The reaction mixture was afterwards incubated on a Thermocycler (2720 Thermal Cycler, Applied Biosystems, California, USA) using the following program:
Initial denaturation 94°C 5 min Denaturation 94°C 30 sec
Annealing 60°C 30 sec
Elongation 72°C 30 sec
Final elongation 72°C 7 min
Storage 8°C ∞
2.14.2 Amplification of DNA fragments for molecular cloning/ Touchdown PCR
A specific pair of primers, defining the DNA-region of interest designated for cloning into the pGEM®-T Easy vector (Promega, Wisconsin, USA), was designed and purchased. DNA fragments selected for eventual cloning were amplified with the Phusion™ High-Fidelity DNA polymerase (Finnzymes, Finnland). This polymerase encompasses not only the 5´ 3´ DNA polymerase activity but also a 3´ 5´ exonuclease (proofreading) activity. The following PCR reaction mixture was used:
30x
1 µl cDNA
5 µl 5x Phusion™ HF buffer
2.5 µl dNTPs (2 mM)
0.5 µl Primer fw (10 pmol/µl) 0.5 µl Primer rev (10 pmol/µl)
0.75 µl MgCl2 (50 mM)
0.7 µl Phusion™ High-Fidelity DNA-Polymerase (0.02 U/µl) 14.05 µl ddH2O
To increase specificity, sensitivity and yield, a touchdown PCR program was applied. The initial annealing temperature will be above the projected melting temperature (Tm) of the used primers, then it progressively transitions to a lower, more permissive annealing temperature over the course of successive cycles (Korbie and Mattick 2008). The primer will anneal at the highest temperature which is least-permissive of nonspecific binding. The generated fragments will be further amplified during subsequent rounds at lower temperatures and will out-compete the nonspecific sequences to which the primers may bind at those lower temperatures (Don et al.
1991, Hecker and Roux 1996).
The reaction was carried out in the Primus 25 advanced Thermocycler from Peqlab (Erlangen, Germany). The following Touchdown PCR program was used:
Initial denaturation 98°C 30 sec
Denaturation 98°C 10 sec
Annealing 65°C 30 sec -1°C
Elongation 72°C 3 min
Denaturation 98°C 10 sec
Annealing 58°C 30 sec
Elongation 72°C 3 min
Storage 8°C ∞
2.14.3 Colony PCR
Colony PCR is a method used to examine many different clones in a short period of time. In this study, it was conducted to identify clones from an agar plate after ligation and transformation procedure, which have integrated the desired PCR product. The following reaction was used:
30x 10x
1 µl Primer fw (10 pmol/µl) 1 µl Primer rev (10 pmol/µl) 2.5 µl dNTPs (2 mM) 5 µl 5x buffer
0.75 µl MgCl2
0.5 µl Mango Taq-Polymerase 14.25 µl ddH2O
The reaction mixture was inoculated with a single clone picked from the agar plate with a pipette tip. As a backup, the pipette tip with the clone was also smeared on a fresh and sterile agar plate containing the appropriate antibiotic. The mixture was incubated on a thermocycler (2720 Thermal Cycler, Applied Biosystems, California, USA) using the following program:
Initial denaturation 95°C 5 min Denaturation 95°C 30 sec
Annealing 60°C 30 sec
Elongation 72°C 50 sec
Final elongation 72°C 7 min
Storage 8°C ∞
2.14.4 Genotyping PCR
For the mouse genotyping PCR, DNA had to be isolated first from the tail biopsy. Therefore, the DirectPCR® Lysis Reagent Tail from Peqlab (Erlangen, Germany) was used. 200 µl of the lysis reagent were added to the biopsy as well as 6 µl Proteinase K (10mg/ml). For lysis, the reaction was incubated overnight at 55°C and 600 rpm on a thermomixer. The following day the reaction was incubated for 45 min at 55°C for heat inactivation and then transferred on ice and used for the following PCR reaction:
30x
1 µl DNA
5 µl 5x buffer
2.5 µl dNTPs (2 mM)
0.5 µl Primer fw (10 pmol/µl) 0.5 µl Primer rev (10 pmol/µl)
0.75 µl MgCl2 (50 mM)
0.7 µl Mango Taq DNA-Polymerase (1 U/µl) 14.05 µl ddH2O
The reaction mixture was afterwards incubated on a Thermocycler (2720 Thermal Cycler, Applied Biosystems, California, USA) using the following program:
Initial denaturation 95°C 5 min Denaturation 95°C 30 sec
Annealing 61°C 1 min
Elongation 72°C 1 min
Final elongation 72°C 10 min
Storage 8°C ∞
2.14.5 Reverse Transcription
The reverse transcriptase used in the reverse transcription (RT) PCR transcribes RNA into cDNA. The principle of reverse transcription is, that the added oligo(dT)-primers anneal to the poly(A)-sequences of mRNAs. Corresponding to the standard PCR, the reverse transcriptase can elongate the strand to synthesize cDNA. For the generation of cDNA from total RNA the reverse transcriptase SuperScript II from Life Technologies (Darmstadt, Germany) was used.
Eventually, the cDNA was subject to quantitative real-time PCR analysis. The reaction was performed in three following steps:
1. Step: 1 – 5 µg RNA
Added to a 6 µl volume with ddH2O + 0.5 µl oligo(dT)-primer (0.5 µg/ µl) + 0.5 µl dNTPs (10 mM)
Incubation: 10 min at 65°C 35x
2. Step: + 2 µl 5x First Strand Buffer + 1 µl DTT (0.1 M)
Incubation: 2 min at 42°C
3. Step: + 0.25 µl SuperScript II + 0.75 µl ddH2O
Incubation for reverse transcription: 50 min at 42°C Heat-inactivation: 15 min at 75°C
Storage: 8°C ∞
The cDNA was diluted 1:10 with ddH2O. Before the generated cDNA was subjected to quantitative real-time PCR analyses, the quality of the reverse transcription was analyzed by standard PCR (Section 2.14.1) using specific primers for the housekeeping genes GAPDH or HPRT, depending on the source of the RNA.
2.14.6 Quantitative real-time PCR
Basically, the quantitative real-time PCR relies on the principal of the conventional PCR, but additionally, it enables the quantification of the PCR product during the PCR. This is possible due to the measurement of a fluorescent dye which binds to double stranded DNA and thereby increases its fluorescent signal (Pfaffl 2001, Ponchel et al. 2003). Thus, the increase in the measured fluorescent signal correlates to the amount of generated DNA. The fluorescent dye used in this study was PCR Mastermix Platinum® SYBR® Green qPCR SuperMix-UDG with Rox (Life Technologies, Darmstadt, Germany). Double stranded DNA bound with SYBR® Green can be excited using light of 480 nm wavelength resulting in an emission spectrum with a maximum at 520 nm. The Rox reference dye serves for normalization of non-PCR-related fluctuations in fluorescence. Using the ABI Prism 7900T Sequence Detection System the increase in the fluorescent intensity was measured after every PCR cycle and hence a graph was generated. During the exponential phase of the PCR reaction, in which the conditions are optimal by means of optimal polymerase activity and sufficient amount of reaction materials (primers, MgCl2), the threshold value is determined. This value defines the PCR cycle with the optimal conditions and it is used for further quantification calculations (Ct value). The generated data was evaluated with the Sequence Detection System software (SDS Version 2.1, PE Applied Biosystems). Usually, the used cDNA was diluted 1:20 with ddH2O prior to the quantitative real-time PCR. The reaction for one well of a 396- well plate consisted of the following components:
2.5 μl cDNA
2.5 μl Primer (fw + rev 100 pmol/µl) 5.0 μl SYBR® Green
The relative expression for one sample was measured in duplicates of which the mean value was used for the final calculations. The following program was used on the ABI Prism 7900T Sequence Detection system:
50°C 2 min
Taq activation: 95°C 3 min
Denaturation: 94°C 15 sec
Annealing: 60°C 30 sec
Elongation: 72°C 30 sec
95°C 15 sec
60°C 15 sec
Melting curve 60°C - 95°C 2°C/min
After completion of the run, the data was evaluated with the SDS program and transferred to MS Excel (Microsoft) for further calculations. The relative expression was determined by ΔΔ-Ct-method for which the following formulae were used:
ΔCt = Ct (Gene of interest) – Ct (housekeeping gene) ΔΔCt = ΔCt (Control) – ΔCt (sample of interest) Relative expression = 2ΔΔCt
The mRNA expression of two housekeeping genes, including TBP (TATA box binding protein), PBGD (porphobilinogen deaminase), LDHA (Lactate dehydrogenase A) or HPRT (Hypoxanthine-guanine phosphoribosyltransferase) for human samples was used as a reference. For murine samples Hprt (hypoxanthine-guaninephosphoribosyltransferase) und Tbp (TATA box binding protein) were used for normalization.
2.14.7 Sequence analysis
The standard, non-radioactive sequence analysis according to Sanger is based on the principle of chain termination (Sanger and Coulson 1975). During the sequence reaction not only deoxy-
40x
nucleosides, but also dideoxynucleotides that have been labeled with different fluorescent dyes, will be incorporated into the DNA during the elongation step. Since the dideoxynucleotides do not possess a 3´- hydroxyl group no further nucleotides can be added by the polymerase resulting in a chain termination. Due to the detected dye the nucleotide can be determined and due to the length of the fragments to position of the nucleotides within the DNA strand could be determined. The used reaction consisted of the following components in a total volume of 10µl:
1 µl DNA 2 µl 5x Buffer 1 µl BigDye®
1 µl sequence-specific primer, either the fw or rev (10 pmol/µl) 5 µl H2O
The reaction was incubated on a Thermocycler (2720 Thermal Cycler, Applied Biosystems, California, USA) using the following program:
Initial denaturation 95°C 1 min
Denaturation 95°C 30 sec
Annealing 60°C 2.5 min
Elongation 60°C 5 min
Storage 8°C ∞
When the reaction was completed, 10 µl ddH2O were added to the sample. Gel electrophoresis by specific capillaries, which detect the fluorescent signals of the dideoxynucleotides, was performed in the automatic sequencer 3500XL (Applied Biosystems, Life Technologies, Darmstadt, Germany).