Abstract
Resorbable bioceramics are attractive for medical applications such as bone substitution. Biochemical analysis on cells cultured on these biomaterials is vital to predict the impact of the materials in vivo and RNA extraction is an essential step in gene expression study using RT-qPCR. In this study, we describe simple modifications to the TRIzol® RNA extraction protocol widely used in biology and these allow high-yield extraction of RNA from cells on resorbable calcium phosphates. Without the modifications, RNA is trapped in the co-precipitated calcium compounds, rendering TRIzol® extraction method infeasible. Among the modifications, the use of extra TRIzol® to dilute the lysate before the RNA precipitation step is critical for extraction of RNA from porous ?-tricalcium phosphate (?-TCP) discs. We also investigate the rationale behind the undesirable precipitation so as to provide clues about the modifications required for other resorbable materials with high application potential in bone tissue engineering.
Highlights
Resorbable bioceramics with relatively high solubility are attractive materials to be used as bone substitutes since the implanted materials can eventually be replaced by bone newly formed in vivo [1,2,3,4]
Biochemical analysis on cells cultured on these biomaterials is vital to predict the impact of the materials in vivo and RNA extraction is an essential step in gene expression study using RT-qPCR
We describe simple modifications to the TRIzol® RNA extraction protocol widely used in biology and these allow high-yield extraction of RNA from cells on resorbable calcium phosphates
Summary
Resorbable bioceramics with relatively high solubility are attractive materials to be used as bone substitutes since the implanted materials can eventually be replaced by bone newly formed in vivo [1,2,3,4]. TRIzol®, TRI Reagent® and TRIsureTM are commonly used commercial products from different companies for RNA extraction They are modified versions of the single-step acid guanidium thiocyanatephenol- chloroform extraction technique developed by Chomczynski and Sacchi, which is useful for processing of large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples [5,6]. This method cannot be applied to resorbable calcium phosphate ceramics directly as ions dissolved from the ceramics would co-precipitate with the RNA in the alcohol precipitation step, which prevents the RNA from being re-dissolved into aqueous solution in subsequent steps. Higher RNA concentration is attainable using this method even for the same yield of RNA compared with silica gel based methods as the pelleted RNA can be dissolved in a small volume (10 μL) of buffer while a relatively large volume (100 μL) of buffer
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