Quantifying Cell-Specific Gene Expression

Total RNA was extracted from CD4⁺ TCs, Møs, SMCs and ECs using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Total RNA was reverse transcribed to cDNA using a high-capacity cDNA reverse transcription kit (cat. no. 4368813; Thermo Fisher Scientific, Inc.) at 92˚C for 5 min according to the manufacturer's protocol. Subsequently, qPCR was performed using LightCycler^® 480 SYBR-Green Master Mix (Roche Diagnostics). The following thermocycling conditions were used for the PCR: 35 cycles at 92˚C for 30 sec and 58˚C for 40 sec, and 72˚C for 35 sec. The primer pairs used for qPCR were validated in previous studies, and were as follows: TSP-2, forward: 5'-TGAGTTCCAGGGCACACCA-3'; reverse: 5'-GGCTTTCTGGGCAATGGTA-3'; Bax, forward: 5'-TTGCTGATGGCAACTTCAAT-3'; reverse: 5'-GATCAGCTCGGGCACTTTAG-3'; Bcl2, forward: 5'-CAGAAGATCATGCCGTCCTT-3'; reverse: 5'-CTTTCTGCTTTTTATTTCATGAGG-3' (24 (link),25 (link)). mRNA levels were normalized to the internal reference gene GAPDH levels using the 2^-^ΔΔ^Cq method (26 (link)).

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Qi L., Wu K., Shi S., Ji Q., Miao H, & Bin Q. (2020). Thrombospondin-2 is upregulated in patients with aortic dissection and enhances angiotensin II-induced smooth muscle cell apoptosis. Experimental and Therapeutic Medicine, 20(6), 150.

Publication 2020

TRIzol® reagent LightCycler® 480 SYBR-Green Master Mix

Bcl2 Cdna Diagnostics Gapdh Gene Mrna Primer Reverse transcription Sybr green Trizol Tsp 2

Corresponding Organization :

Other organizations : People's Liberation Army No. 150 Hospital, Chinese People's Liberation Army, Beijing Anzhen Hospital, Capital Medical University

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Variable analysis

independent variables

Total RNA extraction method (TRIzol reagent)
Reverse transcription method (high-capacity cDNA reverse transcription kit)
QPCR method (LightCycler 480 SYBR-Green Master Mix)
Thermocycling conditions (35 cycles at 92°C for 30 sec, 58°C for 40 sec, and 72°C for 35 sec)

dependent variables

MRNA levels of TSP-2, Bax, and Bcl2

control variables

Reference gene (GAPDH) for normalization of mRNA levels

controls

Positive control: None specified
Negative control: None specified

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