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

. 2024 Apr 29;121(19):e2315168121. doi: 10.1073/pnas.2315168121

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

Copyright © 2024 the Author(s). Published by PNAS.

This article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

PMC Copyright notice

Fig. 2. — NICHE for quantifying target mRNA in living cells. (A) A schematic diagram of DNAT probes for detecting three target mRNA. T1, T2, T3, and T4 constitute the structural skeleton of the DNAT probe. The 5′ end of T1, T2, and T3 are partially complementary to P1, P2, and P3, respectively. Target 1, GAPDH mRNA. Target 2, PD-L1 mRNA. Target 3, CK mRNA. (B) The specificity of DNAT probes for detection of three target mRNAs. FAM, Cy3, and Cy5 represent the fluorescence signals from P1, P2, and P3, respectively. FI, fluorescent intensity. (C) A cross-sectional view of NEED for delivering DNAT probes into living CTCs. (D) The delivery efficiency of NEED and the viability of cells after probe delivery. DNAT probes labeled with FAM dye (without quencher) are delivered into cells for calculating the delivery efficiency. Propidium iodide (PI) dye is used to evaluate cell viability. The statistical data are obtained by counting 1,000 cells/chip from three independent assays. (E) Fluorescent characterization of probe delivery and cell viability on a compact single-cell trap array (the distance between the two cells is 10 μm). DAPI, blue dye for cell nucleus. Calcein AM, green dye, is used to evaluate cell viability. DNAT probe is labeled with Cy5 (without quencher). (Scale bar, 50 μm.) (F) Delivery dose of DNAT probes in HCC827 cells (10,000 cells) by NEED, liposome-based delivery, and simple incubation with free probe, using fluorescence intensity obtained via flow cytometry. (G) Fluorescent images of cells detected by DNAT probes. (Scale bar, 5 μm.) (H) Ratios of fluorescent intensity from 300 cells detected in three separated experiments. Error bars represent the SD. (I) Relative quantification of RNAs detected by qPCR. ΔCt is the difference between the Ct value of PD-L1 RNA and the Ct value of GAPDH RNA. The value of ΔCt is negatively correlated with target RNA concentration. PD-L1 is not expressed in 293FT cells, and the Ct value of PD-L1 is set at 40. ***P-value < 0.001. NS, no significant difference. Error bars in (B), (D), (F), and (I) represent the SD of three independent experiments.