3. METHODS
3.1. DNA Techniques
3.1.11. Real-time qPCR
Real-time quantitative PCR (qPCR) is a nucleic acid amplification method based on the principal of PCR that allows relative or absolute quantification of template DNA. The amplified DNA can be detected during amplification in real-time by measuring the intercalation of a fluorescence dye.
Expression analysis can be further performed with real-time reverse transcriptase PCR (RT-PCR), for which transcription of RNA into cDNA is neccessary. A more detailed explanation of this procedure is described in 3.1.11.3.
3.1.11.1. Real-time qPCR with SYBR Green
The asymmetrical cyanine dye SYBR Green, was used for these experiments unless otherwise noted.
After intercalation of SYBR Green into dsDNA, the resulting DNA-dye-complex absorbs blue light (λmax = 488 nm) and emits green light (λmax = 522 nm). The PCR can be divided into three phases. The first phase is characterized by the limitation of template DNA and the fluorescence intensity is not detectable above the background signal. In the second phase, DNA is exponentially amplified and can be quantified, since the fluorescence intensity is proportional to the amont of PCR product.
Fluorescence is then measured at the end of elongation. The third phase ends in a plateau where the fluorescence intensity of SYBR Green is at its maximum. The cycle, where the fluorescence is for the first time significantly higher than the background, is termed Cycle Threshold (CT).
Primers were carefully chosen to avoid primer dimers and side products. For mRNA analysis, amplicons should include introns and primers should be boundary spanning to exclude the amplification of genomic DNA.
Primers were designed according to the following criteria: They had a similar melting temperature of about 60 °C, with a length of 19-22 nt and a GC content of 40-60%. The desired length of the amplified PCR product was between 80-300 bp. Primers were furthermore optimized by the determination of the optimal annealing temperature in a gradient PCR and analysis of the amplified products on an agarose gel.
SYBR Green intercalates all dsDNA. Therefore a melting curve analysis is performed at the end of the real-time PCR to evaluate for the presence of primer dimers or side products among the real product.
This is achieved by constantly increasing the temperature, allowing the DNA strands to dissociate depending on their specific melting point. The fluorescence of SYBR Green then gradually decreases, since it cannot intercalate dsDNA anymore.
For every primer pair a standard curve was generated to calculate the PCR efficiency to allow for an adequate determination of the relative amounts of unknown templates. Templates of higher
concentration (e.g. a transcript positive cell line) were serially diluted 5 times 1:10 and duplicates were measured to create a standard curve. The slope of the standard curve displays the efficiency of the PCR with these primer pairs. A slope of 1 represents a duplication of PCR product in each cycle and displays a perfect reaction. This standard curve was used to calculate the relative or absolute amount of template in a sample of interest by loading the curve into the same run. The absolute quantification is only possible, if the exact copy number or amount of a control is known.
A no template control and for expression analysis a -RT (cDNA synthesis reaction without reverse transcriptase) control were included into each run, to prove the absence of DNA contamination.
Table 3-11 Real-time SYBR Green Reaction
Reagents Amount
fw Primer [10 μM] 1 μl
rev Primer [10 μM] 1 μl
template DNA 10 μl
cDNA 1.5 μl
DEPC-H2O 7.3 μl
Table 3-12 Real-time SYBR Green Program
Temperature Time Cycles
95 °C 7 min 1
95 °C 10 s
55 °C 30 s
72 °C 7 s
45
3.1.11.2. Real-time qPCR with TaqMan probes
More specificity can be gained using TaqMan probes for quantification of transcripts. In this work TaqMan probes were used for quantification of miRNA expression levels. The function of this type of probes is based on the Förster Resonance Energy Transfer (FRET). The probe contains a quencher (e.g. Dabcyl) on one end and a reporter fluorescence dye on the other (e.g. Fam, Vic). Additionally to its polymerase activity, the Taq polymerase harbors a 5’-3’ exonuclease activity allowing the degradation of the probe from its 5’-end during complementary strand synthesis. This leads to a separation of quencher and reporter fluorescence dye and finally the loss of the FRET signal.
Fluorescence was measured at the end of elongation.
The quantification of the amount of a template of interest was calculated using an intra assay control (e.g. a housekeeping gene) for normalization and the corresponding standard curve for the transcript.
As general reference GAPDH from cDNA or genomic DNA was used for measurement of mRNA transcripts or DNA, respectively. The amount of miRNAs was related to the endogenous miR-21.
Using different TaqMan probes with different fluorescence dyes allows for the measurement of multiple templates in one PCR (multiplex PCR). Components of the reaction mixture and the program are listed in tables 3-13 and 3-14.
Methods
Table 3-13 Real-time Reaction with TaqMan Probes
Reagents Amount
fw Primer 1 μl
rev Primer 1 μl
TaqMan probe 0.4 μl
Mix 10 μl
DEPC-H2O to 18.5 μl
cDNA 1.5 μl
Table 3-14 Real-time Program with TaqMan Probes
Temperature Time Cycles
95 °C 7 min 1
95 °C 10 s
55 °C 30 s
72 ° 7 s
45
The real-time qPCR was performed in duplicate on the Rotor-Gene 6000™. Subsequent analysis was performed using the Rotor-Gene 6000™ software.
3.1.11.3. Reverse-Transcriptase PCR (RT-PCR)
To analyze RNA transcripts in a PCR reaction, the RNA has to be reverse transcribed to cDNA. The cDNA synthesis was done with the SuperScriptIII Reverse Transcriptase according to the manufacturer’s instructions. If procurable, 1 μg RNA was initially used. Both random primers and specific primers were used for the analysis of mRNA transcripts and stem-loop primers for the quantification of miRNA transcripts (see 3.1.11.4). The conditions used for the reverse transcription of mRNA are shown in table 3-15. First, RNA dNTPs and primers were mixed and heated to 65 °C for 5 min, then incubated on ice for 1 min and afterwards the remaining components were added. The synthesis of cDNA was accomplished by incubation at 50 or 55 °C for 1 h for random or specific primers, respectively. Synthesis was followed by inactivation of the reverse transcriptase at 70 °C for 15 min and incubation on ice priot further usage or storage. The program for using stem-loop primers is given in chapter 3.1.11.4.
Table 3-15 SuperScriptIII Revere Transcription Reaction
Reagents Amount
RNA 1 μg
dNTPs [10 mM] 0.5 μl stem-loop primer [1 μM]
or random primer [250 ng/μl]
or specific rev primer [2 μM] 1 μl 5x first strand-buffer 4 μl
DTT [0.1 M] 2 μl
RNaseOUT [40 units/μl] 0.1 μl SuperScript III RT [200 units/μl] 0.25-1 μl
DEPC-H2O to 20 μl
After cDNA synthesis 0.4 μl RNAse H [200 units/μl] was added and the mixture was incubated at 37
°C for 20 min. RNAse H was inactivated by heating at 65 °C for 10 min and tubes were put on ice prior to use or stored in aliquots at -20 °C. Obtained cDNA was then directly used in real-time PCR applications or stored in aliquots at -20 °C.
3.1.11.4. Real-time Stem-loop PCR
A recently established method for the detection and quantification of miRNAs was described in 2005 (Chen et al., 2005a). In this method, a stem-loop primer is used for cDNA synthesis and afterwards the transcripts are measured by real-time PCR using TaqMan probes. Different stem loop primers harbor an equal 5’-end, which affords formation of a stem loop and differ in the last 6 nucleotides at their 3’-end, which is complementary to the 3’ end of a specific miRNA. This method allows detection of very small amounts of mature miRNAs (~ 25 pg). Figure 3-1 shows a schematic overview.
Figure 3-1 Schematic Overview of Stem-loop RT-PCR
cDNA Synthesis is performed using specific primers binding to the last 4-6 nt of a miRNA and fold into a stem-loop structure at the 5’ end. Real-time PCR is performed using a specific primer for the miRNA and a universal reverse primer, binding to a region within the loop of the stem-loop primer. A TaqMan probe binding specifically to the miRNA can be used in real-time PCR to determine the relative amount of the amplified product (Christine Henning).
For detection of miRNAs expressed from the BHRF or BART clusters of EBV, exemplarily the miRNA BHRF1-3 and miRNA BART-5 were used, respectively.
Total RNA (1 μg) was used for the cDNA synthesis with the corresponding stem loop primers. A stem-loop primer for miR-21 and a reverse primer for GAPDH were included as internal controls.
cDNA synthesis was performed as described by Varkonyi-Gasic (Varkonyi-Gasic et al., 2007).
First, dNTPS were mixed with DEPC-H2O and 1 pmole of each stem-loop primer and 2 pmole of GAPDH reverse primer. The mixture was heated to 65 °C for 5 min and incubated on ice for 2 min to allow primer binding. Then the remaining reagents listed in table 3-16 were added.
Methods
Table 3-16 Reverse Transcription Reaction for Stem-loop Primers
Reagents Amount
First strand buffer [5x] 4 μl
DTT [0.1 M] 2 μl
RNAseOut [40 U/μl] 0.5 μl
Superscript III RT [1 U/μl] 0.25 μl
DEPC-H2O to 19 μl
Subsequently 1 μl RNA was added and a pulsed RT reaction was performed, shown in table 3-17.
Table 3-17 Reverse Transcription Program for Stem-loop Primers
Temperature Time Cycles
16 °C 30 min 1
30 °C 30 s
42 °C 30 s
50 °C 1 s
60
4 °C ∞
After cDNA synthesis 0.4 μl RNAse H [200 units/μl] was added and the mixture was incubated at 37
°C for 20 min. RNAse H was inactivated by heating at 65 °C for 10 min and tubes were put on ice prior to use or stored in aliquots at -20 °C.