TRIzol reagent (Invitrogen) or mirVana PARIS System (Ambion, Texas, USA) were respectively employed to isolate total or serum RNA. Subsequently, RNA quantitation and quality determination were performed on the Nanodrop 2000 spectrophotometer platform (Thermo Fisher Scientific, United States). Thereafter, TaqMan miRNA (miR-375 and U6 snRNA) probes (Applied Biosystems, USA) were utilized to perform RT-qPCR according to the manufacturer’s protocol (26 (link)) for mature microRNAs or mRNA using the SYBR Green (Applied Biosystems) method. RT-qPCR was carried out on the 7900 detection system (Applied Biosystems), with U6 small nuclear RNA (snRNA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serving as normalization standards for miRNA and mRNA, respectively. A comparative threshold cycle method was used and values were computed using the 2−ΔΔCt equation. Each reaction in this experiment was run three separate times. The qRT-PCR primer sequences are listed in Table 1 .
Protocol detail
Quantitative Analysis of miRNA Expression
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Glyceraldehyde 3 phosphate dehydrogenase
Mirna
Mrna
Primer
Serum
Sybr green
Trizol
U6 small nuclear rna
Corresponding Organization :
Other organizations : Lishui City People's Hospital, Southeast University, First Affiliated Hospital of Wannan Medical College, Wannan Medical College
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Variable analysis
independent variables
- Isolation method for total or serum RNA (TRIzol reagent or mirVana PARIS System)
- Expression levels of miR-375 and U6 snRNA (measured by RT-qPCR)
- RNA quantitation and quality determination using Nanodrop 2000 spectrophotometer
- Normalization standards for miRNA (U6 snRNA) and mRNA (GAPDH)
- Three separate runs for each RT-qPCR reaction
- Positive control: None specified
- Negative control: None specified
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