Quantitative real-time polymerase chain reaction (qPCR)

A real-time polymerase chain reaction (qPCR) is a laboratory technique in molecular biology which allows to detect and quantify RNA. Similar to traditional PCR (end-point detection), the idea of qPCR is to amplify a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. Nevertheless, unlike conventional PCR, real-time PCR allows monitoring of the desired product at any time in the 𝐸 ∗= 𝐸′+ 𝑖𝐸′′ 𝐸′ =𝐼𝑓𝑜 𝜀𝑜 cos(𝛿) 𝐸′′ = 𝐼𝑓𝑜 𝜀𝑜 sin(𝛿) 𝑡𝑎𝑛𝛿 = 𝐸′′ 𝐸′

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amplification process. For traditional PCR data is collected at the end-point (plateau), while real-time PCR collects data in the exponential growth phase. By using real-time PCR post PCR detection methods like electrophoresis in agarose, which are not precise and time consuming, can be avoided [25].

Another variant of PCR is a reverse transcription PCR (RT-PCR), which can be combined with real-time PCR (RT-qPCR) and such a combined technique was used in this study. In RT-PCR, RNA is used as a template to synthesize complementary DNA (cDNA) by enzyme called reverse transcriptase, which further serves as the template in the real-time PCR. To obtain cDNA in RT reaction a few components are needed: purified extracted RNA of interest, deoxyribonucleotides (dNTPs) as building blocks of cDNA, reverse transcriptase, non-specific primers and RNase inhibitor [26].

Two common methods for the detection of PCR products in real-time PCR are: (1) non-specific fluorescent dyes that intercalate with any double-stranded DNA, and (2) sequence-specific DNA probes consisting of oligonucleotides that are labelled with a fluorescent reporter which permits detection only after hybridization of the probe with its complementary sequence [27]. In the first option, commonly used fluorescent dye is a SYBR Green and in the second method, applied in the study of this thesis, is a technique with usage of TaqMan Probes. TaqMan probes are oligonucleotides having a fluorescent probe (reporter) attached to the 5' end and a quencher to the 3' end. During PCR amplification, these probes anneal to the target sequences of cDNA between the forward and reverse primers and as polymerase replicates the template with TaqMan bound, it also cleaves the fluorescent probe due to polymerase 5'- nuclease activity. Thereby, the distance between the reporter and the quencher increases causing the transfer of energy; the fluorescent emissions of the reporter increase captured by the Sequence Detection instrument and displayed by the software [25].

The PCR process normally is composed of approximately 25-50 cycles and contains a few main steps: the first (denaturation), at around 95 °C, allows to disrupt DNA template into single- stranded DNA molecules; the second (annealing), at a temperature of around 50-60 °C, allows the binding of the primers with the DNA template; the third (extension/elongation), at between 68 - 72 °C, facilitates the polymerization carried out by the DNA polymerase [26].

In this work, gene expression analysis has been performed by firstly reverse transcription carried out at different temperature as follow:

1) 25 °C for 10 min; 2) 37 °C for 2 h; 3) 85 °C for 5 min;

45 Activation of Taq

polymerase enzyme

4) 4°C for ∞.

Then, qPCR (Taqman assay) could be performed using two pre-steps (1 and 2) and proper qPCR reaction composed of 40 cycles (Step 3 and 4):

1) 50 °C for 2 min; 2) 95 °C for 10 min;

3) Denaturation at 95 °C for 15 s;

4) Annealing/Extension at 60 °C for 1 min [25].

The machine for both RT and qPCR was supplied by Applied Biosytems, USA. After the reactions, gene expression can be analysed by absolute or relative quantitation. Absolute quantification relates the PCR signal to input copy number using a calibration curve, while relative quantification measures the relative change in mRNA expression levels correlated to mRNA expression of endogenous control (reference gene). Housekeeping genes e.g. glyceraldehyde 3-phosphate dehydrogenase or β-actin can be used as a reference gene [27].

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PART III