Quantitative real-time Taqman PCR and Fluidigm Dynamic Array

sequence during sequential amplification cycles. In real-time PCR reactions, the buffer in which the reaction takes place is added with a fluorescent dye that changes its fluorescence emission in function of the level of the amplification achieved for each single cycle, that is in real-time as opposite to the classic end-point PCR where the amplification level is assessed only at the end of the reaction. Several different real-time PCR techniques exist, whit specific up and

down sides. In this thesis, I have used the TaqMan real-time PCR system. In this method in the master mix are present not only two sets of primers that define the sequence of cDNA that will be amplified, but also a probe complementary to a cDNA sequence located in the region defined by the primer pair. The probe is conjugated with a fluorescent dye and a fluorescence quencher. Moreover, the enzyme used in this reaction is a modified version of the Taq polymerase used in classic PCR: the TaqMan enzyme not only polymerases oligonucleotide sequences complementary to the template cDNA, but also has an exonuclease activity that allows the hydrolysis of the probe bound to the cDNA. For each cycle of amplification then, the TaqMan enzyme starts the amplification at the primer-annealing site and during the synthesis of the complementary strand it encounters and digests the probe. The quencher is thus released from the fluorescent dye and fluorescence can be measured. The real-time PCR was run in duplicates in 384-well plates (Applied Biosystems) with 10 ng/well initial amount of cDNA. The probes used were bought conjugated with the FAM dye from a commercial producer (LifeTechnologies) and used with a commercial master mix (Applied Biosystems). The reaction took place in an ABI Prism 7900HT machine and data were collected and analysed with the SDS software suite (Applied Biosystems). The thermal cycling conditions were as follows: 2 minutes at 50˚C, 10 minutes at 95˚C, 45 cycles of a denaturation step at 95˚C for 15 seconds and an annealing/extension step at 60˚C for one minute. A calibrator sample was used for every plate and consisted of either spleen, Peyer’s patches or SG from a control animal.

To relative quantification was obtained using the comparative threshold cycle (Ct) method. The Ct cycle of the endogenous control (normally mammalian 18S rRNA) was used to normalise for the initial cDNA amount of each sample by subtracting from the average between Ct duplicates of the gene of interest the average of the endogenous control, obtaining in this way the ΔCt value. To calculate the ΔΔCt value, the ΔCt of each sample was subtracted to the chosen reference sample (usually PP or SG from a control animal). The ΔΔCt value for each sample was then use to calculate the relative quantification (RQ value)

using the equation RQ=2-ΔΔCt_{. The number two in the equation represents the}

doubling of the amplification product for every cycle of PCR, considering an optimal efficiency of the PCR. For TaqMan PCR, this approximation refers to a high efficiency reaction where the fluorescence doubles for each cycle of amplification. In order to confirm the PCR efficiency, in initial set-up experiments

serial 25 _{dilutions (i.e. 1:1, 1:32; 1:1,032) of a control cDNA tissue positive for}

the gene of interest (i.e. spleen cDNA) were used and the observed CT values reflected the expected difference of 5 cycles of amplification for each sample dilution.

For the real-time PCR analysis of FACS-sorted cells a different technique was used that allowed the relative quantitation of gene expression starting from small amounts of initial mRNA. The sorted cDNA samples, extracted with the RNeasy micro kit and retrotranscribed with the Thermoscript kit as described in the above sections, were loaded on a 48x48 Fluidigm Dynamic Array integrated fluidic circuits (IFCs) chip together with the real-time master-mix (Applied Biosystems) and TaqMan probes (Lifetechnologies) described above. Microfluidic chips allow the use of nano-volumes of cDNA, master mix and

probes in comparison with the micro-volumes necessary for the 384-wells plate method described above. The system can load up to 48 different samples requiring as little as 5 µl of each sample at 5 ng/µl concentration to run as many as 48 different assays (45 genes of interest and 3 housekeeping genes). Any chip can thus run 2’304 single real-time reactions at a time. The Fluidigm chip, loaded with samples and reagents, was then run on a Fluidigm Biomark HD system. As calibrating sample, I used the same Peyer’s patches sample used for the 384-wells plate system and the three housekeeping genes used were eukaryotic 18s, β-actin and hypoxanthine-guanine phosphoribosyltransferase (HPRT). The PCR reaction started with a cycle of 10 minutes at 95˚C, followed by 40 cycles formed by a 95˚C for 15 seconds denaturation step followed by a 60˚C for one-minute annealing/elongation step. Relative quantitation was

calculated using the 2-ΔΔCt method described above.

A table with the list of all the primers and probes used for this thesis is reported in Table 3.3.

Gene Product mRNA Accession

Number Assay ID Source

Mouse AID NM_009645 Mm00507774_m1 Applied Biosystems

Mouse

CXCL13 NM_018866 Mm00444533_m1 Applied Biosystems

Mouse CXCR5 NM_007551 Mm00432086_m1 Applied Biosystems

Mouse CCL19 NM_011888 Mm00839967_g1 Applied Biosystems

Mouse CCR7 NM_007719 Mm00432608_m1 Applied Biosystems

Mouse BAFF NM_033622 Mm00446347_m1 Applied Biosystems

Mouse IL-4 NM_021283 Mm00445259_m1 Applied Biosystems

Mouse IL-21 NM_021782 Mm00517640_m1 Applied Biosystems

Mouse Ltβ NM_008518 Mm00434774_g1 Applied Biosystems

Mouse LtβR NM_010736 Mm00440235_m1 Applied Biosystems

Mouse CCL5 NM_013653 Mm01302427_m1 Applied Biosystems

Mouse CCL7 NM_013654 Mm00443113_m1 Applied Biosystems

Mouse CCR1 NM_009912 Mm00438260_s1 Applied Biosystems

Mouse HPRT NM_013556 Mm00446968_m1 Applied Biosystems

Mouse β-actin NM_007393 4352341E Applied Biosystems

Eukaryotic 18S X03205.1 4319413E Applied Biosystems