Veriquest sybr green quantitative pcr master mix

Total RNA was isolated from SCG axons, parental cell soma, or from cytosolic and mitochondrial fractions using Direct-Zol™ RNA MiniPrep (Zymo Research) according to the manufacturer’s instructions. For reverse transcription, equal amounts of purified RNA was mixed with cDNA SuperMix (Quanta Biosciences™) and incubated in a thermal cycler (MJ Research) for 5 min at 25 °C, followed by 30 min at 42 °C and 5 min at 85 °C. qRT-PCR analysis was performed with gene specific QuantiTect primers (Qiagen) for COXIV or β-actin. Gene specific primers for rat pre-miR-338,-204,-185, and -134 were designed using the OligoPerfect Designer (Life Technologies). The COXII and U6 snRNA primers used in the study were identical to those reported previously [13 (link)]. Quantitative PCR was performed using VeriQuest SYBR Green quantitative PCR Master Mix (Affymetrix) and a StepOne Real-Time PCR system (Applied Biosystems). For each experimental RNA sample, the PCR reactions were run in triplicate. The results were analyzed using the StepOne™ Software (Applied Biosystems). Primer sequence list (sense strand, 5′ to 3′):

Total RNA was isolated from SCG axons or parental soma using Direct-Zol™ RNA MiniPrep (Zymo Research) according to the manufacturer’s instructions. For reverse transcription, equal amounts of purified RNA were mixed with cDNA SuperMix (Quanta Biosciences™) and incubated in a Thermal Cycler (Life Technologies) for 5 min at 25 °C, followed by 30 min at 42 °C and 5 min at 85 °C. qRT-PCR analysis was performed with gene specific QuantiTect® primers (Qiagen) using VeriQuest SYBR Green Quantitative PCR Master Mix (Affymetrix). For each experimental RNA sample, the PCR reactions were run in triplicate, using a StepOne™ Real-Time PCR system (Applied Biosystems). The results were analyzed using the StepOne™ Software (Applied Biosystems), normalizing target mRNA levels to ribosomal protein subunit 18 (RPS18) or GAPDH mRNA levels.