Quantification of Let-7 MicroRNA Levels

Two steps of Real-time PCR were performed on 20 ng of transcribed total RNA using the TaqMan MicroRNA Reverse Transcription kit and TaqMan^® Universal PCR Master Mix II, without uracil N-glycosylase (Thermo Fisher Scientific), according to the manufacturer’s instructions. TaqMan microRNA assays (assay ID): hsa-let-7a (000377), hsa-let-7b (002619), hsa-let-7c (000379), hsa-let-7d (002283), hsa-let-7e (002406), hsa-let-7f (000382), hsa-let-7g (002282), hsa-let-7i (002221), hsa-miR-98 (000577), and cel-miR-39-3p (000200) were used in the assay. All PCR reactions were performed on the 7900HT system (Applied Biosystems, Foster City, CA, USA) under the following conditions: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The relative expression level of each Let-7 member was determined using the comparative CT method, which was defined as 2^−ΔCt, where ΔCt = Ct of the Let-7 member—Ct of cel-39 as described previously [11 (link)].

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Tsai Y.S., Huang C.I., Tsai P.C., Yeh M.L., Huang C.F., Hsieh M.H., Liu T.W., Lin Y.H., Liang P.C., Lin Z.Y., Chen S.C., Huang J.F., Chuang W.L., Dai C.Y, & Yu M.L. (2022). Circulating Let-7 Family Members as Non-Invasive Biomarkers for Predicting Hepatocellular Carcinoma Risk after Antiviral Treatment among Chronic Hepatitis C Patients. Cancers, 14(8), 2023.

Publication 2022

TaqMan MicroRNA Reverse Transcription kit TaqMan® Universal PCR Master Mix II uracil N-glycosylase 7900HT system

Assay Microrna Real time pcr Reverse transcription Uracil n glycosylase

Corresponding Organization : Kaohsiung Medical University

Other organizations : National Pingtung University of Science and Technology

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Variable analysis

independent variables

TaqMan MicroRNA Reverse Transcription kit
TaqMan® Universal PCR Master Mix II, without uracil N-glycosylase (Thermo Fisher Scientific)

dependent variables

Relative expression level of each Let-7 member

control variables

20 ng of transcribed total RNA
TaqMan microRNA assays (assay ID): hsa-let-7a (000377), hsa-let-7b (002619), hsa-let-7c (000379), hsa-let-7d (002283), hsa-let-7e (002406), hsa-let-7f (000382), hsa-let-7g (002282), hsa-let-7i (002221), hsa-miR-98 (000577), and cel-miR-39-3p (000200)
PCR conditions: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min
7900HT system (Applied Biosystems, Foster City, CA, USA)

positive control

Cel-miR-39-3p (000200)

negative control

Not specified

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