Ncbi primer blast

Real-time quantitative PCR (qPCR) was performed by Roche LightCycler® 480 using LightCycler® 480 SYBR Green I Master Kit (Roche GmbH, Germany). Primers for PGRN (GRN) were designed using the NCBI Primer Blast and purchased from Eurofins MWG Operon GmbH, Ebersberg, Germany. Primers and probes for PGRN were GRN-forward (5′-CAAATGGCCCACAACACTGA-3′), GRN-reverse(5′-CCCTGAGACGGTAAAGATGCA-3′), and GRN-probe (5′-6FAMCCACTGCTCTGCCGGCCACTCMGBNFQ-3′). Real-time qPCR experiments were run under 58 °C annealing conditions and amplification was run for 45 cycles. A melting curve was implemented in each experiment to prove single product amplification. Data were analyzed using ΔCt calculations where GAPDH or GUSB served as housekeeper control for normalization. The amplification efficiency was experimentally assessed or assumed as 2 (doubling each cycle). Relative mRNA expression levels compared to housekeeper gene expression (efficiency-ΔCt) were used for visualization.

The RNA from the cells stored in RNAlater^TM was extracted using a Quick-RNA Microprep Kit (R1051, Zymo Research, Freiburg, Germany), according to the manufacturer’s protocol. The RNA was eluted in 13 µL nuclease-free water, and the concentration was measured using the NanoDrop^TM 2000 (Thermo Scientific, Germany) by spectrophotometry. The RNA was used for cDNA synthesis using the RevertAid RT Kit (K1691, Thermo Fisher Scientific, Germany). Primers were designed using NCBI Primer-BLAST purchased from Eurofins Genomics (Ebersberg bei München, Germany). The primer list is provided in Table 1. qPCR was performed using PerfeCTa SYBR^® Green FastMix (95072, Quantabio, VWR, Darmstadt, Germany) and the CFX Opus 96 real-time PCR detection system (Bio-Rad, Feldkirchen, Germany). The input cDNA corresponded to 1 ng of total RNA, and the reaction involved an initial denaturation/activation step at 95 °C for 3 min, followed by 40 cycles of 95 °C for 20 s and 60 °C for 50 s. A melt curve analysis was performed at the end to confirm the melting temperature of the PCR product. The cycle threshold (Ct) values were determined from the software output (CFX Maestro, Bio-Rad, Germany) and were used to calculate the fold changes in gene expression using the ΔΔCt method [43 (link)]:

Whole RNA was extracted using the EZNA Total RNA kit (Omega Bio-Tek) and quantified using microspectrophotometry (NanoDrop One; Thermo Fisher). cDNA was prepared using the High-Capacity cDNA reverse transcription kit (Applied Biosystems). PCR primers (Table S3) were designed using the UCSC genome browser in silico PCR and NCBI Primer-BLAST and manufactured commercially (Eurofins Genomics). Quantitation was performed by the threshold cycle (2^−ΔΔCT) method with expression calculated relative to housekeeping gene control (actin) and untreated condition (PBS).