Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2018 Jul 23;8:11038. doi: 10.1038/s41598-018-29350-5

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

PMC Copyright notice

Experimental strategy and example data. (A) Schematic depicting sites of Cas9/sgRNA cleavage relative to ALK and EML4 genes. Chevrons depict direction of transcription. Cleavage sites are within introns. Exon (ex) numbers are shown. P1 and P2 are primers for Taqman PCR assays. Taqman hybridization probe is shown with fluorophore (green) and quencher (black) that separate upon hybridization to amplicon. (B) Reconstruction experiment showing linearity of Taqman qPCR assay. (C) Experimental timeline showing irradiation, recovery, Cas9/sgRNA challenge, and harvesting of DNA for analysis. (D,E) Example Taqman PCR data. Independent cultures were irradiated, challenged, and DNA collected for analysis. Amplification curves are shown for single-copy internal standard (RNase P, Panel D) or for EML4-ALK junctions (Panel E). Irradiation was performed at an LET of 108 keV/μm). Parallel cultures not challenged with Cas9/sgRNAs were included in the experiment in Panel E but showed no detectable amplification products after 40 cycles of amplification. Plots depict normalized fluorescence reporter values (ΔRn) as a function of cycle number. Green line denotes software-determined threshold for determination of amplification (C_t) values.