Total RNA was extracted using the TRIZOL reagent (TAKARA, Kusatsu, Japan). First-strand cDNA was generated from 1-2 µg of total RNA using a commercial SuperScript II First Strand Synthesis System (TAKARA, Kusatsu, Japan). Real-time PCR was performed using SYBR Green PCR Master Mix (TAKARA, Kusatsu, Japan) in a Step One Plus RT-PCR system (Roche, Basel, Switzerland). The amplified transcript level of each specific gene was normalized to β-actin using the 2−ΔΔCt method. The sequences of primers used in this study are listed in the Supplementary Materials (Table S2) .
Superscript 2 first strand synthesis system
Manufactured by Takara Bio
Sourced in Japan
The SuperScript II First Strand Synthesis System is a laboratory equipment product designed for the synthesis of first-strand cDNA from RNA templates. It provides a reliable and efficient method for the conversion of RNA into complementary DNA (cDNA) for use in various molecular biology applications.
Automatically generated - may contain errors
2 protocols using superscript 2 first strand synthesis system
1
Quantitative Gene Expression Analysis
2
Silencing LN-α5 expression in HTR8/SVneo cells
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The LN-α5 siRNA sequences (5′-GCATCAGCTTCGACAGTCA-3′) and scramble RNA were synthesized by Ribobio (Guangzhou, China). HTR8/SVneo cells were transiently transfected with either LN-α5 siRNA or scramble RNA as a control by using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer's protocol. The transfection efficiency was further assessed by western blot. qPCR qPCR was performed as previously described [19] . Briefly, total RNA was isolated from cells or tissues by the use of Trizol (Takara Biotechnology Co. China), and then reverse-transcription to cDNA was performed by the use of Superscript II First-Strand Synthesis System (Takara Biotechnology, Japan). Primers are listed in Table 2.
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