Total RNA from HuR-NP- and C-NP-treated cells and untreated control cells were isolated using Trizol reagent (Life Technologies, Grand Island, NY, USA), and the RNA quality was determined using Denovix DS11 spectrophotometer. Complementary DNA (cDNA) was synthesized from 1 µg RNA/sample using a QuantScript cDNA synthesis kit (Bio-Rad, Richmond, CA, USA). An amount of 3 µg of synthesized cDNA, quantified using Denovix DS11 spectrophotometer, was subjected to real-time quantitative reverse transcriptase (qRT)-PCR (Bio-Rad CFX96™TouchReal-Time PCR Detection System; Richmond, CA, USA) using the premix iQ SYBR green qRT-PCR kit (Bio-Rad) as previously described [30 (link),41 (link)]. The oligonucleotide primers (Integrated DNA Technology, Coralville, IA, USA) and their sequences for the amplification of HuR, BCL-2, and 18S RNA are shown below.
The PCR cycling parameters and all other experimental conditions followed have previously been described [41 (link)]. The cycle threshold (Ct) value assessed by qRT-PCR was calculated for the transcripts and was normalized to a housekeeping gene. The changes in mRNA expression levels were expressed as fold change relative to control. Each sample was run in triplicate. The experiments were repeated at least three times for reproducibility and subjected to statistical analysis.
The PCR cycling parameters and all other experimental conditions followed have previously been described [41 (link)]. The cycle threshold (Ct) value assessed by qRT-PCR was calculated for the transcripts and was normalized to a housekeeping gene. The changes in mRNA expression levels were expressed as fold change relative to control. Each sample was run in triplicate. The experiments were repeated at least three times for reproducibility and subjected to statistical analysis.
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