Power sybr green pcr master mix

Total RNA was extracted with TRIzol reagent and reverse transcribed into cDNA. qPCR was done using Power SYBR Green PCR Master Mix (TIANGEN, Beijing, China). Primer sequences of GAPDH, PRL-3 and stathmin for qPCR were the following: PRL-3 Forward: 5′-GCTTCCTCATCACCCACAAC-3′, Reverse: 5′-ACTTC ACACACACGCACCAC-3′; stathmin Forward: 5'-TCAG CCCTCGGTCAAAAGAAT-3′, Reverse: 5′-TTCTCGTG CTCTCGTTTCTCA-3′; GAPDH Forward: 5′-TCTCTGC TCCTCCTGTTC-3′, Reverse: 5′-GCCCAATACGACCA AATCC-3′. The comparative 2^-ΔΔCt-method was used for relative quantification, with GAPDH as endogenous reference.

Total RNA of spleen tissue was extracted using RNA simple Total RNA Kit (TIANGEN Biotech Co., Ltd., Beijing, China), and was reversed transcription as cDNA by FastKing gDNA Dispelling RT Supermix (TIANGEN Biotech Co., Ltd., Beijing, China) after genomic DNA was removed. Reverse transcription was performed at 42 °C for 15 min. qPCR was performed by ABI Prism 7700 sequence detection system (Applied Biosystems, Thermo Fisher Scientific, Inc., New York, NY, USA) and the reaction mix containing 1 μL cDNA, forward and reverse primers (1 µL each), 10 µL Power SYBR Green PCR Master Mix (TIANGEN Biotech Co., Ltd., Beijing, China) were fixed to a final volume of 20 µL. PCR reactions were performed as follows: initial denaturation at 95 °C for 15 min, followed by 40 cycles of denaturation at 95 °C for 10 s, annealing at 62 °C for 32 s and extension at 72 °C for 30 s, followed by a final extension at 72 °C for 10 min. β-actin was used as internal control the of mRNA expression, and the comparative 2^−ΔΔCq method was employed to quantify relative gene expression. The primers used in this study are listed in Table 2.