Quantitative real-time polymerase chain reaction (qRT-PCR) amplification
This protocol is extracted from research article:
LncRNAs Landscape in the patients of primary gout by microarray analysis
PLoS One, Feb 18, 2021; DOI: 10.1371/journal.pone.0232918

Four significantly more highly expressed lncRNAs (TCONS_00004393, ENST00000566457, NR_003542, and ENST00000430770) and four significantly more lowly expressed lncRNAs (NR_026756, TCONS_00003286, TCONS_00017125, and NR_104125) were randomly selected from the microarray results to detect their expression levels in all 96 samples (including 12 samples previously used for microarray experiment). The information of eight lncRNAs is listed in S1 Table. Extracted total RNA was reverse-transcribed to cDNA with random primer using SYBR Green RT reagents (Bio-Rad, USA). In brief, the RT reaction was performed for 60 min at 37°C followed by 60 min at 42°C, using oligo (dT) and random hexamers. PCR amplifications were performed by using SYBR Green Universal Master Mix. These reactions were performed in duplicate containing 2×concentrated Universal Master Mix, 1 μL of template cDNA, and 100 nM of primers in a final volume 12.5 μL, followed by analysis in a 96-well optical reaction plate (Bio-Rad). All reactions were performed in triplicate. The PCR results were quantified by the 2-ΔΔct method against β-actin and GAPDH for normalization. The primer sequences used in the validation of lncRNAs in this paper were are listed in Table 2.

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