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Primer testing indicated that transcripts with reads per kilobase per million mapped reads values greater than 3 at any of the two time points were suitable for qPCR-based gene expression analysis.
We next randomly selected one FlyPrimerBank primer pair for each of 326 randomly selected genes expressed in 0- to 4-hr-old Drosophila embryos, on the basis of the aforementioned criteria, for experimental qPCR primer testing.
For primer testing and identity confirmation, the fragments from preliminary PCR were cloned and sequenced.
Primer testing and sequencing resulted in 88 gene sequences (Table 2).
Six individuals of P. gonoacantha were screened during primer testing, resulting in amplicons for 15 primer pairs (Table 1).
Initial primer testing included 12 samples of B. integrifolia from three populations in the Hunter Valley region, Australia (Appendix 1).
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Genomic DNA derived from human placenta was used to generate standard curves for each primer tested.
Out of 208 primers tested, 20 were found to be polymorphic.
Only 5.2% of the primers tested produced clear amplicons in all 16 species.
For polymorphism assessment, 27 mulberry genotypes were checked and 10 of the primers tested were shown to be polymorphic.
Of several primers tested, Primer-01 (Operon Kit F) produced a profile that differentiated the population with 88% (P<0.001) efficiency based on multivariate logistic regression analysis of banding profiles.
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