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

Our RNA extraction method involved modification of several steps in the conventional TRIzol protocol to make it compatible with the pulverization step and to enhance RNA yield and purity. First, after incorporating the pulverized material into TRIzol, sealed tubes were placed on a rotator to maximize tissue exposure to the action of this reagent. Second, we centrifuged the TRIzol-homogenized tissue mixture before addition of chloroform to prevent carryover of undigested tissues to subsequent extraction steps and, thus, to enhance RNA purity. Third, we added 1.2 M of sodium chloride and 0.8 M of sodium acetate to the aqueous layer prior to RNA precipitation. This is a typical modification to the TRIzol protocol for tissues with a high content of proteoglycan. We thoroughly investigated the importance of this modification in the context of purifying RNA from articular cartilage by comparing our method to two other protocols where the addition of high salt concentration was omitted. In the first protocol we followed the exact steps of our method except for the addition of high salt concentration (Our method). In the second protocol we combined TRIzol and silica-membrane RNA purification columns as follows. We followed the steps of our method until we separated the aqueous layer, to which we added 1 volume of 70% ethanol, loaded the solution on an RNeasy® spin column (Qiagen), and followed the manufacturer's protocol to purify RNA (TRIzol/RNeasy). Both protocols resulted in a significant reduction in RNA purity, which confirms the necessity of using a high concentration of salt to isolate pure RNA from articular cartilage homogenates. Notably, our method purifies cartilage RNA with appropriate purity even though it involves only one phase-separation step; unlike other methods that require two phase-separation steps prior to high salt RNA precipitation. The last modification to the TRIzol protocol was based on previous reports and included washing RNA precipitates twice to remove traces of salt given the high salt concentration used in the precipitation step. It is noteworthy that using more than 100 mg of cartilage tissue in our method significantly reduced RNA yield and purity. If purifying RNA from more than 100 mg of cartilage is desired, we recommend scaling up the volume of TRIzol accordingly, or, alternatively, dividing the tissue into 100 mg portions followed by combining the resulting aliquots of purified RNA.

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Manufacturer protocol

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