Primer design and establishment

The nucleotide database of the National Center for Biotechnology Information (NCBI; https://www.ncbi.nlm.nih.gov/nuccore/) was used to design the required primer pairs. In the case of multiple transcript variants, the one with the longest nucleotide sequence was chosen to take into account all other variants of the target gene. To ensure a high specificity of the primers, a PCR product length of 90–300 base pairs (bp) was chosen. The maximum melting temperature difference of the primers was minimized to ensure the most effective amplification of the desired sequence of the target gene by both the forward primer and the reverse primer. Subsequently, the “intron inclusion” function was activated. This function was used to ensure that forward and reverse primers were separated by at least one intron of the corresponding genomic DNA sequence. In the case of several transcript variants of the target gene, the range of the primers to be synthesized was additionally limited to that sequence which includes all transcript variants. The proposed primer variants for the desired target gene were then checked for temperature ranges of possible loop formation using the software GeneRunner (HelioGenetics, New Jersey, USA). Only those primer pairs were selected as sufficient where this temperature range and the annealing temperature of the primers were far apart. The primers were manufactured at Eurofins (Ebersberg, Germany). The determination of the optimal annealing temperatures of the selected primer pairs was done by gradient PCR. This was also used to exclude possible by-products during the amplification process. The primer sequences are shown in Table 1.

Primer sequences and annealing temperatures (X) used for mRNA analysis

GCATATGCTTGTCTCAAAGA /

CCAAAGGAACCATAACTGAT

AGCCCTCTCAGTGTCTACGC /

CTCCTTGGTGATGCTTCCAT

TCACGAGGACACCCTTAGCA /

GGCTTCAGGGTGGAGAACAA

TTCTTCTTTGTCCTGCCGCT /

GAAGGCGCGCTTGAACTC

TGCTGACCAAGAATAAGGCCC /

AATGGCATAGGCTTGGTTCGT

AAGCATTCGTCCAGCAGC /

AGAGGTTTCATGCGCAGC

GAGCTCCTGGTCCACACTG /

TAGAACGACCTCCCAGGCA

AACGAGATGGACAAGAACCGATGT /

GACCGAGGTCATCAGACTTTTGGA

TGCTTGCTTCATCCCGTTCA /

ACTTCCCGTCTCTGCTTTAGG

Cycling conditions: 95 °C, 3 min (one cycle) / 95 °C, 30 sec + X °C, 20 sec + 72 °C, 20 sec (45 cycles) / 72 °C, 5 min (one cycle) / 95 °C, 2 min (one cycle)