Methodological strategies for transgene copy number quantification in goats (Capra hircus) using real‐time PCR

Abstract

Taking into account the importance of goats as transgenic models, as well as the rarity of copy number (CN) studies in farm animals, the present work aimed to evaluate methodological strategies for accurate and precise transgene CN quantification in goats using quantitative polymerase chain reaction (qPCR). Mouse and goat lines transgenic for human granulocyte-colony stimulating factor were used. After selecting the best genomic DNA extraction method to be applied in mouse and goat samples, intra-assay variations, accuracy and precision of CN quantifications were assessed. The optimized conditions were submitted to mathematical strategies and used to quantify CN in goat lines. The findings were as follows: validation of qPCR conditions is required, and amplification efficiency is the most important. Absolute and relative quantifications are able to produce similar results. For normalized absolute quantification, the same plasmid fragment used to generate goat lines must be mixed with wild-type goat genomic DNA, allowing the choice of an endogenous reference gene for data normalization. For relative quantifications, a resin-based genomic DNA extraction method is strongly recommended when using mouse tail tips as calibrators to avoid tissue-specific inhibitors. Efficient qPCR amplifications (≥95%) allow reliable CN measurements with SYBR technology. TaqMan must be used with caution in goats if the nucleotide sequence of the endogenous reference gene is not yet well understood. Adhering to these general guidelines can result in more exact CN determination in goats. Even when working under nonoptimal circumstances, if assays are performed that respect the minimum qPCR requirements, good estimations of transgene CN can be achieved.

Challenges for the Characterization of Genetically Modified Animals by the qPCR Technique in the Era of Genomic Editing

“…To overcome this limitation, commercial kits DNA binding to silica-based matrices, followed by elution, can be used to remove inhibitors and organic solvents, such as chloroform. According to Burkhart et al [34] and our experiments [28] , gDNA obtained by silica matrix-based methods are more efficient for amplification by qPCR. An inhibition test using either internal controls or evaluation of the linearity of the calibration curves should be performed to determine the suitability of the extracted DNA for real-time PCR amplification [28,30] .…”

“…For real-time PCR use, RNA is removed from the nucleic acid preparation with enzymes, as RNase A and RNase T1. However, the prior work of our group demonstrated that this DNA extraction method compromises the efficiency of qPCR [28] . According to published reports [29,30] , phenol/chloroform extracted DNA needs further purification to be used for real-time PCR.…”

Heterologous Biosynthesis of the Fungal Sesquiterpene Trichodermol in Saccharomyces cerevisiae

“…qPCR is a rapid, sensitive, and accurate technique to quantify the copies of transgenes ( Batista et al, 2014 ). qPCR results showed that one or two copies of FgTRI5 , two or three copies of optimized FgTRI5 , six or seven copies of TaTRI4 , and one or two copies of TaTRI11 were integrated into respective strains ( Supplementary Figure 6 ).…”

Genetic characteristics of polycistronic system‑mediated randomly‑inserted multi‑transgenes in miniature pigs and mice

“…The single standard curve-absolute method [with internal reference (representative of the genomic copy number)] is an efficient method for the detection of transgene copy numbers ( 7 , 20 ). Fluorescent quantitative polymerase chain reaction (qPCR) analysis is used to draw a log a N-ΔCq absolute quantification standard curve (N indicates the copy number; ΔCq represents the difference in the fluorescence threshold between the exogenous gene and the reference gene; and ΔCq=Cq transgene -Cq internal ) ( 21 ).…”