Qtower 2.2 real time pcr system

To detect the expression level of miRNA in bamboo, stem-loop qRT-PCR was employed in this study. Primers were designed specifically based on individual miRNA using previous method [14] (link). U6 snRNA was selected as internal control [15] (link). More detailed information of primers was supported in File S1.
On the basis of leaf samples in moso bamboo and ma bamboo, cDNAs were synthesized from total RNA with the miRNA-specific stem-loop RT primer according to reference [16] (link). Subsequently, qRT-PCR was carried out using a SYBR Green I Master Kit (Roche, Germany) on a QTOWER2.2 Real-Time PCR System (Analytik Jena). The final volume was 10 µl, containing 5.0 µl 2×SYBR Premix Ex Taq, 0.2 µl of each primer (10 µM), 0.8 µl of cDNA and 3.8 µl of nuclease-free water. All reactions were repeated three times and the amplification was conducted as follows: initial denaturation at 95°C for 10 min, followed by 50 cycles at 95°C for 10 s, and 62°C for 10 s. There was three biological experiments. Expression levels were normalized to that of the internal control, and the relative value was calculated using previous method [17] (link).

According to previous studies (Hui et al., 2019 (link); Zeng et al., 2019 (link); Hui et al., 2020b (link)), we used RNAiso Plus (Takara, Japan) to isolate total RNA from GA and testis. Then we reverse-transcribed RNA into cDNA using PrimeScript RT reagent Kit (Takara, Japan). We performed the qPCR using qTower 2.2 real-time PCR system (Analytik Jena, Germany). The primers were synthesized by Sangon Biotech (Shanghai, China) and listed in Supplementary Table 1.

All components were configured for the qTOWER2.2 Real-time PCR System (Analytik Jena AG, Jena, Germany) and centrifuged at 6,000 rpm for 30 s at 4 °C using a PCR plate centrifuge. Then, samples were placed into the quantitative PCR instrument and amplified according to the above procedure. The amplification steps were as follows, the fluorescence quantitative PCR program and system: (a) Step 1, 95℃ for three minutes; (b) Step 2, 95℃ for 10 s; (c) Step 3, 58℃ for 30 s + plate read; (d) Step 4, Go to Step 2, 39 cycles; (e) Step 5, melt curve analysis (60–95℃, + 1℃/cycle, holding time = four seconds).
The nine samples were separated, and three duplicate wells were set up. Then, the relative expression of the target gene in each sample was automatically calculated using the instrument software qPCRsoft 3.2 through the Pfaffl method. Then, ACT2 was used as the internal reference, and the CK group was used as the control group to estimate the relative expression.

Gene expression levels were determined using quantitative real-time polymerase chain reaction (qRT-PCR). The Reverse Transcription Kit was used to reverse-transcribe total RNA into cDNA (R222, Vazyme, Nanjing, China). Using the SYBR Green master mix performed qRT-PCR as follows: one cycle at 95°C for 30 s, 40 cycles at 95°C for 10 s and 60°C for 30 s (Q311, Vazyme, Nanjing, China). The final volume was 10 uL (containing 1 uL of cDNA, 5 uL of SYBR Green mix, 0.2 uL each of both forward and reverse primers and 3.6 uL ddH₂O). Real-time detection of SYBR Green fluorescence was conducted using a qTOWER 2.2 Real-Time PCR System (Analytik Jena, Jena, German). The GAPDH gene was amplified to serve as an internal control. The relative quantification values for critical genes were calculated by the 2^−ΔΔCt method [18 (link)]. The gene-specific primers were designed using Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) (listed in Table S4).

RNA was extracted with TRIzol reagent (Invitrogen, United States). We used qTower 2.2 real-time PCR system (Analytik Jena, Germany) to run qRT-PCR with SYBR Premix Ex Taq II (Takara Bio, Japan). All primers are listed in Table 1. Relative mRNA expression levels were normalized to those of β-actin and calculated by the 2^−ΔΔCt method.

Total RNA was extracted from foxtail millet and Arabidopsis using the TRIzol reagent (Invitrogen, USA). After digestion with DNaseI (Takara, Japan), 3–5 μg of total RNA was prepared, and cDNA was synthesized via reverse transcription using M-MLV Reverse Transcriptase (Promega, USA). Semi-quantitative RT-PCR was performed using 2 × Taq PCR StarMix with Loading Dye (GenStar, China). The PCR conditions were 95°C for 5 min, followed by 25 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s, with a final step at 72°C for 10 min. Quantitative RT-PCR (qRT-PCR) was performed using 2 × Ultra SYBR Mixture (CWBIO, China) on a qTower 2.2 Real-Time PCR System (AnalytikJena, Germany). The PCR conditions were 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, and 60°C for 1 min. Relative gene expression levels were calculated using the 2^−ΔΔCT method (Livak and Schmittgen, 2001 (link)).