Superscript 3 platinum sybr green one step kit

Total RNA was extracted from the middle rosette leaves of AC and NAC plants by using the TRizol reagent. Upon verification of RNA integrity by agarose gel electrophoresis, 5 μg of RNA was treated with DNase RQ1 (Promega, Madison, WI, USA) in a 50 μl reaction and purified with the RNA Clean and Concentrator kit (Zymo Research, Irvine, CA, USA) following the instructions of the manufacturer. Quantitative RT-qPCR was performed with SuperScript III Platinum SYBR Green One-Step kit (Invitrogen) and gene-specific primers (Supplementary Table 1). Relative expression was calculated by the 2^−ΔΔCt method (Pfaffl, 2001 (link)) considering NAC as the reference sample and actin or rRNA 18S as the normalizing transcript. Three independent RNA analyses were performed for each one of two different biological experiments.

MM.1S, MOLP-8, U266.B1, and MM.1S-shHDAC3 cells were grown for 6 hours in presence or absence of 6 μmol/L lactacystin or 60 nmol/L bortezomib. Carfilzomib was added to MM.1S or MOLP-8 cells for 6 hours at a concentration of 20 nmol/L or 15 nmol/L, respectively. Untreated MM.1S-HDAC3 cells, MM.1S-SIAH2 cells, and corresponding control cells were harvested during exponential growth phase. Total RNA was extracted using the RNeasy Plus Mini kit (Qiagen, 74134) according to the manufacturer's protocol. RNA purity was verified by UV absorbance measurements at 260 and 280 nm on a NanoDrop 1000 (Thermo Fisher Scientific). qRT-PCR was performed on the isolated RNA with the SuperScript III Platinum SYBR Green One-Step Kit (Invitrogen, 11746–500) as recommended by the manufacturer, on a Bio-Rad CFX96 real time PCR instrument. Relative mRNA expression was calculated with the comparative C_t method (ΔΔC_t method; ref. 26 (link)) and normalized using GAPDH expression levels as reference.
The following primers were used for qRT-PCR assays: