Abi quantstudio 5 real time pcr instrument

The total RNA from the spleen was extracted using NcmZol reagent (NCM Biotech, China), following the manufacturer's protocol. The nucleic acid spectrophotometer (Eppendorf, Germany) was used to determine the RNA concentration and purity at 260 nm and 280 nm (A260/280 ¼ 1.80 À 2.00). Reverse transcription to complementary DNA (cDNA) was carried out using PrimeScript TM reverse transcription reagent kit with gDNA eraser (Takara Bio Inc, Japan) according to manufacturer's protocol. A total of 20 mL reaction mix, constituting 2 mL of 5x diluted cDNA template, 0.8 mL of each forward and reverse primer, 10 mL of TB Green TM Premix ExTaq TM (Takara Bio Inc, Japan), 0.4 mL of ROX reference Dye II and 6 mL DEPC water was used to perform Real-time PCR was on ABI QuantStudio 5 Real-Time PCR Instrument (Applied Biosystems, ThermoFisher Scientific, United States). Primers used for qRT-PCR were based on published target sequences (Table S1). Primers were normalized against the mRNA level of 18-S as an internal control and the CON þ Saline group was used as calibrator. Thermal cycling was initiated with an initial denaturation stage of 30 s at 95 � C, followed by 40 cycles of 95 � C for 5 s and 60 � C for 30 s, and the dissociation stage. The relative mRNA expression of the target genes was analyzed using the 2 À DDCT method.