Iq5 cycler pcr machine

RNA extraction was performed using a RNA extraction kit (Qiagen) according to the manufacturers’ instructions. 500 ng of RNA was used as template for cDNA preparation with the iScript cDNA Synthesis Kit (Bio-Rad). Quantitative PCR was performed in a IQ5 cycler PCR machine (Bio-Rad), using the Kapa SYBR Fast qPCR kit (Kapa Biosystems). Primers used are described in Supplementary table 1. Expression levels were corrected for expression of two house-keeping genes, Actb and Gapdh, averaged and presented as fold changes.

Total RNA was extracted using a NucleoSpin® RNA kit (MACHEREY-NAGEL, Düren, Germany) and quantified with a Nanodrop ND-1000 (Wilmington, DE, USA). 500 ng of RNA was used as template for cDNA preparation with the reverse transcription system from TAKARA (TAKARA BIO INC). Quantitative real-time PCR (qRT-PCR) reactions were performed with SYBR Green (Applied Biosystems®, Austin, USA) according to the manufacturer's instruction. qRT-PCR was performed in an IQ5 cycler PCR machine (Bio-Rad). The levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA were used as an endogenous reference to normalize the quantities of target mRNA by using the formula 2^−ΔΔCT (ΔΔCT = ΔCT_sample-ΔCT_control). All primer sequences were indicated in Table S1.