TRI Reagent® MRC

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Avoid washing cells before the addition of TRI Reagent as this may contribute to RNA degradation.	- Perform centrifugation for phase separation in the cold ( 4 - 10 C ). If performed at elevated temperatures, a residual amount of DNA may sequester in the aqueous phase.

Upstream tips
- Avoid washing cells before the addition of TRI Reagent as this may contribute to RNA degradation.
Protocol tips
- Perform centrifugation for phase separation in the cold ( 4 - 10 C ). If performed at elevated temperatures, a residual amount of DNA may sequester in the aqueous phase.

Publication protocol

Total RNA was isolated using TRIzol (Invitrogen, Grand Island, USA) according to the manufacturer׳s instructions. cDNA was prepared from 1 to 2 μg of RNA using the Superscript II reverse transcriptase (Invitrogen) with random hexamer primers. Real-time PCR reactions (50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 30 s and 72 °C for 10 min) were performed in triplicate using SYBR Green (Applied Biosystems, Foster City, CA, USA) in Step one plus (Applied Biosystems, Life Technologies, Carlsbad, CA) with primers specific for thymidine kinase, thymidine synthase and dihydrofolate reductase, CDK4, CDK6, CDK2, cyclin D1, cyclin D3, p27, p21, p15, cyclin E1 [Primer sequences are available on request]. The relative gene expressions were normalized to GAPDH using the formula 2−ΔΔCT (ΔΔCT=ΔCTSample−ΔCT Untreated control), where CT is the threshold cycle.

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Manufacturer protocol

Download the product protocol from Molecular Research Center, Inc. for TRI Reagent® MRC below.

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