Site-specific CpG methylation was analyzed in the bisulfite converted cord blood DNA template for PRSS21, and in the placenta DNA template for ANGPT4, PGRMC1 and RGS14, using custom designed bisulfite pyrosequencing assays (Qiagen, USA).
This result is again different from the results of Pérez-Casanova et al. [ 9] who showed, using QPCR with blood cDNA templates, that these genes were differentially expressed between cod at 16°C and at 10°C.
The cDNA was synthesized with the SuperScript™ III kit (Invitrogen, CA USA) using RNA isolated from peripheral blood lymphocytes as template.
PCR was performed with 50 ng of tumor and matched blood DNA as template from the KIT positive cases in a reaction volume of 35 μl, using primers specific for exons 1, 9, 10, 11, 12, 13 and 17 of KIT.
One microlitre of blood-extracted DNA template was used in the primary PCR reaction and 1 μl of the primary reaction was used in the nested reaction.
This might be due to the fact that they used diluted blood samples as templates, and duplicate analysis might solve the problem.
GPR81 was amplified by polymerase chain reaction (PCR) using human fetus kidney cDNA and whole blood genome DNA as template, respectively.
To construct pre-microRNA expression vectors, we first amplified ~0.5 kb DNA fragment covering a pre-microRNA, using genomic DNA from a healthy blood donor as a template.
Site-specific CpG methylation was analyzed in the bisulfite converted cord blood and placenta DNA template for 22 individuals, in duplicate, using a custom-designed methylation bead array platform, following the manufacturers guidelines (Illumina, USA) and as previously described [ 32].
