For a subset of 8 TOIs (5 microarray-identified genes that were qPCR confirmed as > 2-fold differentially expressed between low-quality and high-quality

7 hpf eggs, and 3 IFN pathway genes), expression was also assessed in unfertilized eggs from the same 15 females; two biological replicates (females) were removed from the unfertilized egg qPCR analysis since they had outlier normalizer CT values. Replicate beaker number 2 was used for each female for gene expression analyses. The sequences Ponatinib of all primer pairs used in the qPCR analyses are presented in Table 3. Each primer pair was quality tested to ensure that a single product was amplified (dissociation curve analysis) and that there was no primer-dimer present MK-1775 nmr in the no-template control. Amplicons were electrophoretically separated on 2% agarose gels and compared with a 1 kb plus ladder (Invitrogen/Life Technologies) to ensure that the correct size fragment was being amplified. Amplification efficiencies (Pfaffl, 2001) were calculated using cDNA synthesized from a high quality (female 2) 7 hpf egg RNA sample and from low quality (females 12 and

13) 7 hpf egg RNA samples. For the low quality females, cDNA was synthesized (see method below) from female 12 and 13 RNA samples separately and then pooled. The reported efficiencies (Table 3) are an average of the values for high and low quality females, with two exceptions: discoidin, CUB and LCCL domain containing 1 (dcbld1), and aromatic-L-amino-acid decarboxylase [synonym: dopa decarboxylase (ddc)] amplification efficiencies are reported for the low quality female pool only due to extremely low expression in female 2. Standard curves were generated using either a 5-point 1:3 dilution series starting with cDNA corresponding to

not 50 ng of input total RNA, or a 4-point 1:3 dilution series starting with cDNA corresponding to 16.7 ng of input total RNA [see Table 3 (including footnotes) for details]. First-strand cDNA was synthesized in 20 μL reactions from 1 μg of DNaseI-treated, column-purified total RNA using random primers (250 ng; Invitrogen/Life Technologies) and SuperScript II reverse transcriptase (200 U; Invitrogen/Life Technologies) with the manufacturer’s first strand buffer (1 × final concentration) and DTT (10 mM final concentration) at 42 °C for 50 min. PCR amplification was performed in a 13 μL reaction using 1X Power SYBR Green PCR Master Mix (Applied Biosystems/Life Technologies), 50 nM of both the forward and reverse primers, and cDNA corresponding to 8 ng of input total RNA. The real-time analysis program consisted of 1 cycle of 50 °C for 2 min, 1 cycle of 95 °C for 10 min and 40 cycles of 95 °C for 15 sec and 60 °C for 1 min, with fluorescence detection at the end of each 60 °C step. On each plate, for every sample, the target gene and endogenous control were tested in triplicate and a no-template control was included.