Quantitative Analysis of Lung Immune Markers

Take the upper lobe of the left lung cryopreserved at −80 °C, total RNA was extracted by Trizol (BIO TNT) according to the manufacturer’s instruction, then the total RNA was reverse transcripted into the first strand of cDNA (PrimeScriptTMRT Master Mix (Perfect Real Time) Takara BIO INC) according to the instruction, the concentration and A260/280 of cDNA were checked to judge the purity of the sample extract. Real-time quantitative polymerase chain reaction (qPCR) experiment (TB Green^® Premix Ex TaqTM II (Tli RNaseH Plus) Takara BIO INC) was performed to detect the expression level of IL-33, ST2, IL-4, IL-13, IL-5, TNF-a, IFN-γ (primer sequences are presented in Table 1), and the results were analyzed as previously reported (Wei et al. 2016 (link)).

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Huang X., Yu H., Xie C., Zhou Y.L., Chen M.M., Shi H.L., Tang W.F., Dong J.C, & Luo Q.L. (2022). Louki Zupa decoction attenuates the airway inflammation in acute asthma mice induced by ovalbumin through IL-33/ST2-NF-κB/GSK3β/mTOR signalling pathway. Pharmaceutical Biology, 60(1), 1520-1532.

Publication 2022

PrimeScriptTMRT Master Mix (Perfect Real Time) TB Green® Premix Ex TaqTM II (Tli RNaseH Plus)

Cdna Ifn γ Il 13 Il 33 Lobe lung Primer Real time polymerase chain reaction Tnf a Trizol

Corresponding Organization : Fudan University

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Variable analysis

independent variables

Tissue source: Upper lobe of the left lung

dependent variables

Expression level of IL-33
Expression level of ST2
Expression level of IL-4
Expression level of IL-13
Expression level of IL-5
Expression level of TNF-a
Expression level of IFN-γ

control variables

Cryopreservation temperature: -80 °C
RNA extraction method: Trizol (BIO TNT)
CDNA synthesis method: PrimeScriptTMRT Master Mix (Perfect Real Time) Takara BIO INC
QPCR method: TB Green® Premix Ex TaqTM II (Tli RNaseH Plus) Takara BIO INC

controls

No positive or negative controls were explicitly mentioned.

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