Solvent based methods using commercially available reagents such as Tri-Reagent® (Sigma) or Trizol® (Invitrogen) are simple and adaptable to varying cell numbers, but involve the use of toxic, organic solvents. Column based kits, from suppliers such as Qiagen and Promega, circumvent the use of organic solvents but are far less flexible in terms of increasing cell numbers. If centrifugation of the columns is performed the process becomes tedious for more than a few specimens and cross contamination is more likely. If large numbers of specimens are to be processed the use of vacuum compatible columns is recommended. As with DNA preparation for MRD studies, the final RNA concentration may not be sufficiently high.
DNA contamination is a frequent problem of RNA preparation. Although most applications will design the real-time PCR primers and probes to span exons (some genes are single exons and this is not possible), cDNA synthesis and subsequent PCR may be less efficient and sensitivity reduced due to lower input of cDNA. Removal of contaminating DNA by DNase digestion is easily carried out by utilizing RNase-free DNase followed by the Rneasy Minelute Kit (Qiagen). Our laboratory uses Tri-Reagent® for routine isolation of total RNA from blood and marrow cells.
Specimens for RNA based MRD analysis should be processed as soon as possible to prevent decrease in sensitivity due to RNA degradation. Blood specimens may be drawn directly into PAXgene tubes (Qiagen), however these are expensive, but are of great value as blood specimens may be shipped at room temperature without RNA degradation.
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