Oligo d t 18 mrna primer

Two clusters of floral buds were pooled from 8-week–old plants, snap-frozen in liquid nitrogen, homogenized using a Mixer Mill MM 400 (Retsch) for 30 s with maximum amplitude, and resuspended in 200 μL of TRIzol (Life Technologies). Total RNA was extracted using a Direct-zol RNA Kit (Zymo Research) according to the manufacturer’s instructions, and DNaseI treatment was performed on-column. Total RNA quality and quantity were determined with an Agilent Fragment Analyzer (AATI) using a standard RNA sensitivity kit (DNF-471). Two-hundred nanograms of total RNA samples with RNA Quality Number values >6.0 were used for cDNA synthesis together with the Oligo d(T)₁₈ mRNA Primer (New England Biolabs) and SuperScript III Reverse Transcriptase (Thermo Fisher Scientific). The cDNA was diluted 10× with nuclease-free water, and 2 μL was used as a template for the qRT-PCR. qRT-PCR was performed on a LightCycler 96 Instrument (Roche) using gene-specific and control elF4A primers (Supplemental Table), and Fast SYBR Green Master Mix (Roche). Cycle threshold values were obtained using LightCycler 96 software (Roche), and relative quantification of transcripts (ΔΔCycle threshold values) was performed with an in-house “R” script. For each genotype, 6 to 17 individual first-generation transgenic (T₁) lines were analyzed in technical duplicates.

Cell samples were lysed in 1:1 (v/v) RLT buffer (Qiagen) and 96% ethanol. Total RNA was isolated using the RNeasy Plus Mini Kit (Qiagen) according to the manufacturer’s instructions. RNA was converted into cDNA using Oligo d(T) 18 mRNA Primer (New England BioLabs), dNTP Mix, RiboLock RNAse Inhibitor, RT Buffer, and Maxima Reverse Transcriptase (all from Thermo Scientific). For real-time quantitative PCR (RT-qPCR), 30 ng of cDNA was used with SsoAdvanced Universal SYBR Green Supermix (Bio-Rad). The primers were hTSPO (Bio-Rad, sequence not available), hTBP [31 ], and hRPLP0 [32 (link)]. Data analysis was done from C_t values normalized to the average value of two housekeeping genes (TBP and RPLP0). Results are shown as fold-change expression to Ctrl (delta-delta C_t method).

Cells were harvested in RLT-buffer (Qiagen) after which RNA isolation was carried out using a RNeasy RNA isolation kit (Qiagen) according to manufacturer’s instructions. Purified RNA (1 μg) was converted to cDNA using Oligo-d(T)18 mRNA primers (New England Biolabs) and Maxima RT enzyme according to the manufacturer’s instructions with the addition of dNTPs and a RNAse inhibitor (Thermo Fisher Scientific). cDNA was subjected to quantitative real-time PCR (qRT-PCR) using a DyNAmo HS SYBR Green qPCR kit (Thermo Fisher Scientific) with gene-specific primers (Supplement 2). The samples were run on a Bio-Rad CFX96 qPCR system with an annealing temperature of 60 °C for in total 40 cycles. β-actin was used as a reference gene. The results were analyzed by adjusting the Ct-values to that of β-actin and subsequently comparing the relative expression of receptors within each cell line.