sentence: Quantitative PCRs ( qPCRs ) were conducted using the SsoAdvanced Universal SYBR Green Supermix ( Bio-Rad ) in a CFX96 Touch ™ Real-Time PCR Detection System ( Bio-Rad ) using an initial cycle at 95 ° C for 3 min and 40 cycles consisting of 95 ° C for 10 s , 58 ° C for 20 s , and 72 ° C for 20 s. To assess expression changes of LSU genes , the measured levels were first normalized to ACTIN2 ( ACT2 ) and ELONGATION FACTOR1 ( EF1 ) levels , except for salt and pH 8 treatments where these controls showed strong regulation and where ACT8 and UBIQUITIN10 ( UBQ10 ) were used instead .