Total RNA from the infection area of VIGS plants was extracted using TRIzol reagent, and one microgram of total RNA was reverse transcribed using HiScript II 1st Strand cDNA Synthesis Kit (Vazyme) according to the supplier’s instructions. A SYBR Green-based qRT-PCR was performed on a QuantStudio 7 platform (Thermo Fisher Scientific; Shanghai, China) with a 10 μL reaction system and were used as internal controls. All primer sequences for qRT-PCR are shown in Table S9 Cycle conditions were as follows: pre-denaturation at 95 ℃ for 1 min; 95 °C 10 s; 58 °C 15 s; 72 °C 20 s, 40 cycles, followed by a standard melting curve analysis. The data were processed using the 2−ΔΔCT method [69 (link)].
Quantstudio 7 platform
Manufactured by Thermo Fisher Scientific
The QuantStudio 7 platform is a real-time PCR system designed for high-throughput nucleic acid analysis. It features a 96-well or 384-well block format and supports a variety of chemistries and applications, including gene expression, genotyping, copy number variation, and digital PCR. The instrument provides precise temperature control and optical detection capabilities to enable accurate and reliable data generation.
Automatically generated - may contain errors
2 protocols using quantstudio 7 platform
1
RNA Extraction and qRT-PCR Analysis
2
PD-L1 Copy Number Analysis
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The frozen biopsies from the resected tissue and EBUS-transbronchial needle aspiration samples were analyzed for PD-L1 copy number using the TaqMan Copy Number Assay kit specific to PD-L1 (#Hs03704252). PCR was performed utilizing the TaqMan Genotyping Pro-Amp Master Mix (Thermo Fisher Scientific, Waltham, MA) on the QuantStudio 7 platform (Thermo Fisher Scientific, Waltham, MA). Single-well reactions were run in quadruplicate per sample in a 384-microplate layout following the manufacturer's instructions. In brief, each single-well reaction contained 2 ng of genomic DNA, Master Mix, and PD-L1 TaqMan primer that was duplexed with the TaqMan Copy Number Reference Assay (RNase P). Copy numbers were calculated with the CopyCaller v2.0 Software (Thermo Fisher Scientific, Waltham, MA) using the DDCt relative quantification method, which calculates the relative copy number of a target gene normalized to RNase P. On the basis of the range values within the nonmalignant control tissue, A PD-L1 copy number of 2.5 or greater was defined as a gain of copy number, whereas a PD-L1 gene copy number less than 1.5 was defined as loss of copy number.
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