TRIzol Reagent

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- For low 260/230 readings the best approach is to try washing sample (precipitation) with ethanol to desalt it.

Protocol tips
- For low 260/230 readings the best approach is to try washing sample (precipitation) with ethanol to desalt it.

Publication protocol

Total RNA was extracted with Trizol (Invitrogen), and miRNA was extracted with miRcute miRNA Isolation kit (Tiangen, Shanghai, China). The RNA was converted to cDNA using Primer Script RT Reagent kit (Takara, Tokyo, Japan), which was incubated for 15 min at 37°C followed by 5 min at 85°C. The specific reverse transcription (RT) primer of miR-512-5p was 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGC ACTGGATACGACGAAAGTG-3′. Real-time quantitative PCR was run on a Light-Cycler Roche 480 (Roche Applied Science, Shanghai, China) with reactions consisted of a hot start (10 min at 95°C), followed with 40 cycles of 15 sec at 95°C and 60 sec at 60°C. The expression level of miR-512-5p and p21 was normalized to U6 and GAPDH, respectively. Each sample was run in triplicate to ensure accuracy. The PCR primers for miR-512-5p were 5′-CGGCGGCACTCAGCCTTGAGGG-3′ and 5′-GTGCAGGGTCCGAGGT-3′. The PCR primers for p21 were 5′-CTACTGAGGAGCCAGCGTCTA-3′ and 5′-CTGCCCATCATCATGACCT-3′. The PCR primers for GAPDH were 5′-CTTAGATTTGGTCGTATTGG-3′ and 5′-GAAGATGGTGATGGGATT-3′.

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for TRIzol Reagent below.

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