RNA was isolated from dissected testes, male carcasses, dissected ovaries and female carcasses from the Cas9.1e, Cas9.2a, Cas9.2b, Cas9.2c and Cas9.d transgenic lines in biological triplicates using TRIzol Reagent (Thermo Fisher Scientific) following the manufacturer’s instructions. The total RNA obtained from each tissue was reverse transcribed using the Thermo Scientific Maxima H Minus First Strand cDNA Synthesis Kit with dsDNase according to the manufacturer’s instructions. The real-time quantitative PCR (qPCR) assays were performed using the 7500 Fast Real-Time PCR system. Amplifications were carried out in a solution containing 10 μL 2X Fast SYBR™ Green Master Mix (Thermo Scientific), 2 μL first-stranded cDNA (diluted 1:10) and 800 nM of each primer (for primers see Additional file 9: Table S4), to a final volume of 20 μL. To check reproducibility, each assay was performed with technical triplicates for each of the three biological samples. The Cas9 expression levels were normalized to the housekeeping gene Rpl19 [41] and expression to wild-type samples, and data analysis was performed using the PCR package for R [42] (R scripts and input data are available at https://github.com/genome-traffic/medflyXpaper).

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.