Lightcycler 96 thermocycler

Total RNA was isolated from DLBCL tissues and cells according to the manufacturer's instructions using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Then, the total extracted RNA was used as the template for reverse transcriptional cDNA via Transcript cDNA Synthesis kit (Beijing TransGen Co., Ltd.) according to the manufacturer's protocol. The PCR reaction conditions were initial denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 sec, annealing at 60°C for 30 sec, and extension at 72°C for 30 sec. The primer sequences used were as follows: UCA1 forward, 5′-GCACCCTAGACCCGAAACTT-3′ and reverse, 5′-CCGGACTGCTTCAAGTGTGA-3′; miR-331-3p forward, 5′-GAGCTGAAAGCACTCCCAA-3′ and reverse, 5′-CACACTCTTGATGTTCCAGGA-3′; GAPDH forward, 5′-CCTGACCTGCGTGTGGACT-3′ and reverse, 5′-GCTGTGGATGGGGAGGTGTC-3′; U6 forward, 5′-CGCTTCGGCAGCACATATAC-3′ and reverse, 5′-TTCACGAATTTGCGTGTCAT-3′. GAPDH was used as the endogenous control of UCA1, while U6 was the endogenous control of miR-331-3p. The expression of UCA1 and miR-331-3p were detected by RT-qPCR with SYBP Premix Ex Taq II (Takara Bio, Inc.) and LightCycler^® 96 thermocycler (Roche Diagnostics). The 2^−ΔΔCq (19 (link)) method was used to calculate the experimental results.

EMS58‐1 seeds were obtained by the suppressor screening in smg7‐6 background described in Capitao et al. (2021 (link)). Mutations associated with improved fertility were identified by whole genome sequencing of pooled fertile plants using ArtMAP (Javorka et al., 2019 (link)). The identified cdkd;3‐3 mutation was genotyped by the high‐resolution melting (HRM) qPCR‐based method using primers described in Table S3 using Lightcycler96 thermocycler (Roche) using the inbuilt HRM profile.

Cytokine protein levels in the brain homogenate were quantified using LEGENDplex cytometric bead assays (BioLegend) following the manufacturer’s instructions. Gene transcript levels were determined by qPCR. Total RNA from brain homogenate was achieved using the TRIzol reagent (Life Technologies), which were then converted to cDNA with QuantiTect Reverse Transcription Kit (Qiagen) according to the manufacturer’s instructions. Gene expression was quantified with SYBR Green amplification (Radiant Green master mix; Alkali Science) using a LightCycler 96 thermocycler (Roche). Relative gene expression was then analyzed using the 2-ΔΔCq method relative to housekeeping genes Gapdh and 18S ribosomal RNA.