Validation of differentially expressed genes
About 3 ml of whole blood from 10 initially treated pulmonary TB
patients and 15 healthy people controls (HC) was collected in a heparin
sodium anticoagulant tube. The inclusion criteria of the TB group was
sputum MTB-positive, and/or interferon-gamma release assay
(IGRA)-positive pulmonary TB patients without anti-TB treatment or with
anti-TB treatment for less than 1 month. The inclusion criteria of HC
were IGRA-negative and without abnormal findings in lung computed
tomography (CT) scan. All samples were respectively stimulated with MTB
CFP-10-ESAT-6 fusion protein for 24h(35). Total RNA was extracted using
Trizol reagent (Invitrogen, USA), and cDNA was reverse transcribed from
1μg of purified RNA using PrimeScriptTM RT Reagent Kit
(Takara, Japan). The primer sequences for amplification of the DE genes
were obtained from PrimerBank and synthesized by Sangon Biotech
(Shanghai, China) and shown in Table 1. RT-qPCR amplification of the
genes was performed using the RT-qPCR kit with the SYBRGreen Ⅰ method
(KAPA SYBR® FAST, South Africa). The quantification of
mRNA was examined using RT-qPCR on Roche 480 (Roche, Switzerland) with
the following program: pre-denatured at 95℃ for 3min; 40 cycles of
denaturing at 95℃ for 10s, annealing at 60℃ for the 20s; extension at
72℃ for 1s. RT-qPCR of each cDNA sample was repeated twice, and the
final Ct value was the mean of the two times. The relative expression
amount of Amy2a, Retn, and Sftpd was calculated by the
2−△△Ct method using the GAPDH gene as an internal
reference.