DNA was extracted in duplicate from each mixed meat sample using the same method.
We prepared 2 primer pools for the 7 BWAs of interest, and each pool contained 1 primer pair specific for each BWA. Figure 2 shows the amplification of BWAs in each mixed culture sample using primer pools I and II.
To gain a global overview of the P. cactorum transcriptome and gene activity during the important life stages, we prepared a mixed cDNA sample using RNA from 5 different life stages: mycelia (MY), sporangia (SP), swimming zoospores (ZO), cysts (CY) and germinating cysts with germ tubes (GC).
Pairwise comparisons were performed on randomly paired individual samples from each group labeled respectively with Cy3 or Cy5 or vice versa and using a mixed sample labeled using Cy2 as an internal standard.
The 135 165-bp peak of the pooled cDNAs was purified from the mixed sample by using the Pippin Prep DNA Size Selection System (Sage Science) and confirmed by the Bioanalyzer.
The total RNA was extracted from the mixed sample by using Trizol reagent (Invitrogen, Camarillo, CA, USA), following RNA purification by RNeasy MiniElute Cleanup Kit (Qiagen, Hilden, Germany), according to the manufacture's protocol.
After that, we added 160 µl 5 M NaCl and 530 µl chloroform and vigorously mixed the samples using a vortex mixer before centrifuging.
A reliable marker is an important tool for the broad scale analyses of mixed environmental samples using next-generation sequencing technologies [50], [51], whose importance will probably increase in biodiversity research and conservation biology within the next years [51].
In a previous study using these primers, the detection limits in complex (mixed) DNA samples using 2% agarose gels and ethidium bromide are: 5 pg in a 10 ng mixture (0.05%) for chicken, 0.005% for cow and 0.0005% for pork [33].
Then, the solvent was removed from the mixed lipid samples using rotary evaporator, the obtained dry film being subsequently hydrated using some MilliQ water (Millipore, Bedford MA, USA).
