On the other hand, to optimize the site that mediate maximal knockdown effects, two to three potential target sequences for a given gene should be designed with mFold software and selected based on the predicted secondary structure and ΔG.
Primer pairs were designed with DS Gene software (ver 1.5) (Accelrys)) using publicly available ovine and bovine sequence information (NCBI, http://www.ncbi.nlm.nih.gov/) and the Interactive Bovine In Silico Single Nucleotide Polymorphism (IBISS, http://www.livestockgenomics.csiro.au/ibiss/) systems (Table 2).
A novel, single-tube CGG repeat primed FMR1 PCR technology was designed with two gene-specific primers that flank the triplet repeat region, as well as a third primer that is complementary to the (CGG n repeat.
Primers specific for the target genes were designed with Beacon Designer 3.0 (Premier Biosoft International, Palo Alto, CA, USA) and assays optimised and validated for real-time PCR using SYBR Green chemistry, as described previously [ 84] (also see Additional File 4).
The primers for the genes were designed with Beacon Designer 6.0 software (Premier Biosoft International, Palo Alto, CA), and the stem-loop primers and the forward primers were designed based on the mature miRNA sequences, the reverse primer was the universal reverse primer.
Primers for COQ7 and the housekeeping gene were designed with the Beacon Designer 4 software.
Primers [see Additional file 2: Table S1] for five overlapping fragments of mtDNA D-loop sequence and one Y-chromosome SRY gene fragment were designed with Primer3 [ 42] using mtDNA and SRY gene reference sequences [GenBank:NC001941] and [GenBank:AF026566.1], respectively.
This resistance gene was designed with flanking full frt sites [ 64], which were used to excise the kanamycin gene via induction of FLPe recombinase [ 65, 66].
Reads identified as "matching two locations" did not include those matching rRNA genes as the 10403S pseudochromosome created for this study was designed with only one unique rRNA gene sequence.
Four NBS-LRR genes and one RIN4-like gene were tested and primers for these genes were designed with Primer3 software (http://frodo.wi.mit.edu/primer3/) (see Additional files 6 and 7).
