RNAsimple Total RNA Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- To wipe off RNase, the glassware can be roasted at 150℃ for 4 hours, while plastic can be dipped in 0.5 M NaOH for 10min, washed by RNase-Free ddH2O thoroughly, and sterilized

Upstream tips
- To wipe off RNase, the glassware can be roasted at 150℃ for 4 hours, while plastic can be dipped in 0.5 M NaOH for 10min, washed by RNase-Free ddH2O thoroughly, and sterilized

Publication protocol

Total RNA of placenta from each group was extracted using Trizol and RNA simple Total RNA Kit (Tiangen Biotech, Beijing, China). RNA was quantified by UV absorption measure and 1 µg was reverse transcribed to cDNA. The reverse transcription (RT) was carried out using PrimeScript RT reagent Kit (TaKaRa, Dalian, China) for 15 min at 37°C, 5 sec at 85°C in a 20 µl reaction volume. Real-time PCR was performed using SYBR Premix Ex Taq (TaKaRa) according to the manufacturer’s instruction. Primer sequences were ordered from Sangon (Shanghai, China) (Table 1). The real-time PCR cycling condition for GAPDH, CYP1A1, VEGF-A, VEGF-B, PIGF, Flt-1, fetal liver kinase-1 (Flk-1), soluble Flt-1 (sFlt-1), HIF-1α, Endoglin and IFN-γ is 30 sec at 95°C for incubation, 40 cycles of 15 sec at 95°C and 20 sec at 60°C. To confirm the amplification specificity, the PCR products were subjected to a melting curve analysis. Levels of mRNA expression were analyzed using 2−△△CT method and gene expression was normalized to GAPDH.

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Manufacturer protocol

Download the product protocol from Tiangen for RNAsimple Total RNA Kit below.

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