I searched several protocols for reverse transcription of RNA and all suggest using RNA concentrations of 1ug. Could a lower amount result in inferior transcript representation and therefore decreased quality of the qPCR results?
We use the SensiFAST SYBR Lo-ROX kit from BIOLINE. I was thinking of using 500ng instead of the suggested 1000ng. In order to accommodate the protocol, I would use 0.5ul of RTase to maintain the 1:1 (m/v) ratio. Buffer, DNase, water, and RTmix concentrations for the reaction I would keep the same. For the final dilution of the cDNA, I would dilute 5x instead of 10x to maintain equal concentrations.
Does anyone have experience with this? Does transcript representation suffer? Do the suggested protocol adaptations seem reasonable?
Thanks for the help in advance.