qPCR was performed to detect the localization of known genes (list of used primers is attached in Supplementary Table S1 ). PCR primers were designed using Primer3 (Untergasser et al., 2012 (link)). The expected length of qPCR products was 80–120 bp and the annealing temperature was 60°C. Geneious prime (version 2021.2) was used to increase the specificity of designed primers and to avoid targeting RNA isoforms.
qPCR reaction mix with a total volume of 7 μl contained 2 μl of cDNA, 0.29 μl of forward and reverse primers mix (1:1, 10 μM each), 3.5 μl of 2x TATAA SYBR® GrandMaster® Mix (TATAA Biocenter, Sweden) and 1.21 μl of RNase-free distilled water (ThermoFisher, USA). qPCR was performed using CFX384 Real-Time System (Bio-Rad, USA) as follows: the initial denaturation for 3 min at 95°C, 45 cycles of denaturation for 15 s at 95°C, annealing at 60°C for 20 s and extension at 72°C for 20 s. qPCR melting curves were analyzed to test the reaction specificity. qPCR data were analyzed using workflow published in Sindelka et al., 2008 (link).
qPCR reaction mix with a total volume of 7 μl contained 2 μl of cDNA, 0.29 μl of forward and reverse primers mix (1:1, 10 μM each), 3.5 μl of 2x TATAA SYBR® GrandMaster® Mix (TATAA Biocenter, Sweden) and 1.21 μl of RNase-free distilled water (ThermoFisher, USA). qPCR was performed using CFX384 Real-Time System (Bio-Rad, USA) as follows: the initial denaturation for 3 min at 95°C, 45 cycles of denaturation for 15 s at 95°C, annealing at 60°C for 20 s and extension at 72°C for 20 s. qPCR melting curves were analyzed to test the reaction specificity. qPCR data were analyzed using workflow published in Sindelka et al., 2008 (link).
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