Primescript reverse transcriptase reagent kit with gdna eraser perfect real time

Total RNA was isolated from three biological replicates per treatment, as described above, for preparation and sequencing of the DEG library. Each RNA sample (approximately 1 μg of total RNA) was treated with gDNA Eraser (TaKaRa Bio Inc., Beijing, China) following the manufacturer instructions, to eliminate any contaminant gDNA. The treated RNA solution (10 μL) was reverse transcribed using the PrimeScript™ Reverse Transcriptase Reagent Kit with gDNA Eraser (Perfect Real Time; TaKaRa Bio Inc.) in accordance with the manufacturer’s protocols. Gene-specific primers were designed using Primer 5.0 and are listed in Table S5. GAPDH was used as the internal reference gene. qRT-PCR was performed on the Bio-Rad CFX96™ Real-Time System (Bio-Rad, Hercules, CA, USA) using the SYBR Premix Ex Taq™ Kit (Perfect Real Time; TaKaRa Bio Inc., Beijing, China) in accordance with the manufacturer’s protocols. qRT-PCR conditions were as follows: 30 s at 94 °C for denaturation, then 40 cycles of 5 s at 94 °C, 30 s at 56 °C, and 10 s at 72 °C. The relative expression levels of the target genes were calculated using the 2^−ΔΔCt comparative threshold cycle method. All reactions were conducted on three biological replicates, and the results were analyzed using Bio-Rad CFX Manager software (V1.6.541.1028).

The RNA solution (10 μl), after removal of DNA contamination, was subjected to reverse transcriptase reactions with PrimeScript™ Reverse Transcriptase Reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa) in accordance with the manufacturer’s protocol. Specific primers for 35 miRNAs and 6 target genes were designed using Primer Premier 5.0 (Additional file 1). qRT-PCR was performed using the SYBR Green PCR Master Mix (TaKaRa) on a CFX96 Detection System (Bio-Rad, Hercules, CA, USA). Briefly, the 25 μl PCR reaction contained no more than 100 ng cDNA, 12.5 μl SYBR Premix Dimer Eraser (2×), and 0.3 μM of each primer. The reactions were mixed and incubated at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, 55 °C for 30 s, and 72 °C for 30 s. The expression levels of the miRNAs and target genes were normalized to those of the internal controls U6 [29 (link)] and actin (NCBI, XM_002282480) respectively. The relative expression levels were analyzed using the 2^-ΔΔCT method [30 (link)]. Ct represents the threshold cycle.

The RT-qPCR experiments were used to confirm and analyze the basic expression levels of a subset of candidate genes. Total RNA was isolated from normal green leaf and gold-colored mutant leaf samples collected in May, June and July, as described above. Each RNA sample was treated with gDNA Eraser following the manufacturer’s instructions to eliminate any contaminant gDNA, using 1 μg of total RNA. The treated RNA solution (10 μL) was subjected to reverse transcriptase reactions via PrimeScript™ Reverse Transcriptase Reagent Kit with gDNA Eraser (Perfect Real Time; TaKaRa) in accordance with the manufacturer’s protocol. Gene-specific primers were designed using Primer 5.0 (Supplementary Table S4). The G. biloba gene GAPDH was used as an internal reference gene. RT-qPCR was performed using the CFX96™ Real-Time System (Bio-Rad, Hercules, CA, USA) with the SYBR Premix Ex Taq™ Kit (Perfect Real Time; TaKaRa) in accordance with the manufacturer’s protocol. RT-qPCR conditions were as follows: 30 s at 95 °C for denaturation, followed by 40 cycles of 5 s at 95 °C, 30 s at 55 °C, and 10 s at 72 °C. All reactions were performed in three biological replicates, and the resultant threshold cycle (Ct) values were determined using Bio-Rad CFX Manager software (ver. 1.6.541.1028). Relative expression levels of target genes were calculated with the 2^−ΔΔCt comparative Ct method^{14 (link)}.