RNA was extracted from homogenized cells, tissues or embryos according to the manufacturer's guidelines (TRIzol, Invitrogen). RNA concentration was determined by NanoDrop2000 Spectrophotometer (Thermofisher) and integrity was determined by running 1 µl of RNA and 9 µl 11.1% glycerol on EtBr 1.5% agarose gel in 1 × TAE. cDNA was synthesized from 2 μg of RNA 2 µg of RNA was reverse transcribed to cDNA using Superscript II Reverse Transcriptase with random hexamers and oligo dT primers (all from Invitrogen).
RT-qPCR was performed on a C1000 Touch Thermal Cycler with the CFX96 Optical Reaction Module (Bio-Rad) using 2× KAPA SYBR FAST qPCR mix (Kapa Biosystems) in technical triplicates with gene specific-primers, see [19 (link),26 (link)], following cycling temperatures and times as per manufacturers protocol. ΔCt was determined using reference genes using rpl13α or EF1α as a reference gene and relative expression (RE) levels were compared using the ΔΔCt method.
RT-qPCR was performed on a C1000 Touch Thermal Cycler with the CFX96 Optical Reaction Module (Bio-Rad) using 2× KAPA SYBR FAST qPCR mix (Kapa Biosystems) in technical triplicates with gene specific-primers, see [19 (link),26 (link)], following cycling temperatures and times as per manufacturers protocol. ΔCt was determined using reference genes using rpl13α or EF1α as a reference gene and relative expression (RE) levels were compared using the ΔΔCt method.
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