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 using the TRIzol reagent (Invitrogen). Then, 1 μg of the total RNA was reverse-transcribed, and qRT-PCR was performed using the ABI PRISM 7900 sequence detection system (Biosystems). The PCR amplification was performed in a reaction system containing cDNA, forward and reverse primers, 2X SYBR Green qPCR SuperMix, and distilled water. DKK3 transcript expression was measured by qRT-PCR using the forward primer 5′-TTTTCCACGCAGTTCTTTCC-3′ and the reverse primer 5′-TGAGCCTCTGAGATCCCTGA-3′. For the DKK3 transcript expression measurements, β-actin was amplified as an internal control using the forward primer 5′-CTTAGTTGCGTTACACCCTTTCTTG-3′ and the reverse primer 5′-CTGTCACCTTCACCG TTCCAGTTT-3′. The relative expression of the amplified RNA samples was calculated using the 2−DDCT method. The results are presented as the fold change of each mRNA relative to a control sample (MNCs collected from a healthy volunteer).

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Manufacturer protocol

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