2. CÁLCULOS JUSTIFICATIVOS.
2.2. Fórmulas utilizadas
3.4.1.
Digital PCR
For the allele-specific expression assays, a TaqMan digital PCR (dPCR) was carried out taking advantage of the QuantStudioTM 3D Digital PCR System (Applied Biosystems)
and according to the manufacturer's recommendations. To this end, 15μL of a mix containing cDNA, QuantStudio® 3D Digital PCR Master Mix v2 (2x) and specific TaqMan probes (20x) were loaded into a dPCR chip (QuantStudioTM 3D Digital PCR Chip Kit) with
the help of the QuantStudio 3D Digital PCR Chip Loader, according to the kit's instructions. The amount of sample used for the reaction was calculated and set up in order to ideally have one molecule of cDNA per well. After the chips had been thoroughly sealed, the dPCRs took place in a GeneAmp® PCR System 9700 (Applied Biosystems), with an annealing temperature of 60ºC and 40 cycles of reaction. Lastly, the fluorescence of each well was measured, allowing the absolute quantification of the samples.
The primers and TaqMan probes used for the dPCR were designed with the Custom TaqMan® Assay Design Tool (Thermo Fisher Scientific) and produced by the same company. The FAM probes specifically identified the mutant transcripts, while the VIC probes only recognized the wild type ones. The dPCR was used to analyze the cDNA from the paraffin-embedded tumors for two variants in SETD6 and PTPRT. Tumor cDNA from sporadic CRC patients served as non-carrier control, and cDNA from healthy colon tissue was also used as a control.
3.4.2.
Quantitative real-time PCR
For the quantification of the overall gene expression of two PTPRT downstream target genes (BCL-XL and SOCS3), a quantitative real-time PCR (qPCR) was performed in a 7500 Fast Real-Time PCR System (Applied Biosystems). For this purpose, cDNA was analyzed in a 96-well plate with the TaqMan® Gene Expression Master Mix (Applied
Biosystems) and specific TaqMan probes designed by Thermo Fisher Scientific (BCL-XL assay Hs00236329_m1, SOCS3 Hs02330328_s1). PSMB4 was used as a housekeeping gene whose levels served as a reference (assay Hs00160598_m1), and a pool of 5 healthy colon cDNAs was used as a control. All samples were analyzed in triplicates, and the qPCR reaction took place with an annealing temperature of 60ºC and 40 cycles.
The qPCR results were expressed as a Ct value for each sample-probe combination. The Ct (cycle threshold) is defined as the number of cycles required for the fluorescent signal to exceed the background level. Those measurements differing in more than 1 unit with their corresponding replicates were discarded. The quantification of the relative target gene expression was calculated as 2-ΔΔCt, where ΔΔCt was the difference between
ΔCt of the studied sample and ΔCt of the control pool, and ΔCt was the Ct value of the target gene minus the Ct of the housekeeping gene (PSMB4). The standard deviation was calculated for each sample.
3.4.3.
Promoter methylation assay
Tumor DNA from two PTPRT mutation carriers was used for the promoter methylation assay. DNA obtained from healthy colon or breast was used as a control, as well as tumor DNA from sporadic CRC patients. The first step of this study was the bisulfite conversion of 1μg of DNA, which was done using the EpiTect® Bisulfite Kit (Qiagen) according to its protocol. A methylation specific PCR (MSP) was then performed with the EpiTect® MSP Kit (Qiagen) and using two different sets of specific primers targeting the promoter region of PTPRT. Each set was composed of 2 pairs of primers, which specifically detected methylated (M and M2) or unmethylated DNA (U and U2). These primers had been previously described by Laczmanska et al.177 (in CRC, set 1) and by Peyser at al.178 (in
head and neck carcinoma, set 2) (Appendix, A3).The MSP was carried out following the manufacturer's instructions in a final volume of 25μl, with an annealing temperature of 55ºC and 40 cycles of reaction. Finally, the PCR products were analyzed by electrophoresis in a 2.5% agarose gel, and the fractional methylation was quantified by densitometry using the ImageJ software.
3.4.4.
Pyrosequencing
Pyrosequencing was used for the measurement of PTPRT’s promoter methylation. For this purpose, pyrosequencing primers were designed using the PyroMark Assay Design 2.0 software, consisting in 2 PCR primers targeting a fraction of PTPRT’s promoter (with a biotinylated reverse primer) and a biosynthesis primer targeted between them. All the primers were HPLC purified and are shown in the Appendix, A3. DNA from CRC, healthy colon, breast cancer and healthy breast from PTPRT mutation carriers was obtained, followed by the bisulfite conversion of 1μg of DNA using the EpiTect® Bisulfite Kit (Qiagen) according to its protocol. A PCR was then performed using 1μL of converted product and a Taq polymerase (Thermo Fisher Scientific), with an annealing temperature of 48ºC and 45 cycles of reaction. Commercial high-methylated and non-methylated DNAs were used as controls (CpGenome Human Methylated/Non-Methylated DNA Standard, Millipore).
The sample preparation was performed using 10μL of each PCR product and PyroMark Annealing Buffer, streptavidin-coated Sepharose beads, Binding Buffer, Wash Buffer and Denaturation Solution (Qiagen), following the manufacturer’s instructions and with the help of a vacuum prep work station. Finally, the pyrosequencing took place in a PyroMark Q96 MD sequencer (Biotage, Qiagen) with the help of the Pyro-Q-CpG Software and using PyroMark Gold Q96 reagent Kit (containing the PyroMark Enzyme Mixture, Substrate Mixture and dNTPs for the pyrosequencing reaction) and PyroMark Q96 HS Capillary Tips. The PCRs and subsequent pyrosequencing were performed in triplicates, and the relative methylation (%) and standard deviation were represented.
3.4.5.
Histone binding affinity assay
The histone binding affinity of mutant Pygo1 was tested by isothermal titration calorimetry (ITC) at Mariann Bienz's laboratory (Cambridge University, United Kingdom) as described by Miller et al196. The ITC was carried out at 25°C with an iTC 200
Microcalorimeter (GE Healthcare) following dialysis of purified wild type and mutant His- tagged PHDPygo1-HD1BCL9 and PHDPygo1-HD1B9L complexes in 25mM Tris (pH 8.0) and
100mM NaCl. Titrations consisted of 19 consecutive 2-μl injections of peptide solution (following a pre-injection of 0.5μl) into the protein at time intervals of 120s or 150s. The 15-mer H3K4me2 histone tail peptide was used as previously described194, and its