Controls for realtime PCR

The use of appropriate controls is vital for accurate diagnostic PCR. If absolute quantitation is performed the most convenient standard is a plasmid containing the target sequence. Kits for cloning of PCR products are available from many sources, but some expertise in manipulation of plasmids and bacteria is required. Plasmid standards for many of the common fusion transcripts are available commercially (for example, from Ipsogen, Marseille, France). cDNA produced from in vitro transcribed RNA can also be used to generate standard curves; however it cannot be assumed that the efficiency of reverse transcription is equal to that of the test specimens; also the RNA is subject to degradation.

Plasmid DNA should be isolated using high-quality commercial kits such as those manufactured by Qiagen or Promega. The concentration of the purified DNA should be determined accurately. It is vital that the size (assuming a molecular weight of 660 kD for a base pair) of the plasmid and the inserted sequence are known in order to calculate the number of molecules per microlitre using Avogadro's number (see Chapter 2). As DNA can stick to the walls of test tubes; it is advisable to use carrier DNA. We prepare our plasmid dilutions in a solution of 10 ^g/ml lambda phage DNA in 0.1x TE (Tris-EDTA) buffer. The choice of carrier DNA should be considered carefully to ensure that there is no cross reactivity with PCR primers and probes. The range of standard concentrations should reflect the dynamic range of the assay and the values expected from patient samples; in practice this is often from 106 to less than 10 molecules per microlitre. Standards should be aliquoted and stocks stored at -20°C, working aliquots should be stored at 4°C. These should be discarded when the reproducibil-ity of the lower copy number replicates deteriorates (usually between one to six months).

In addition to positive controls negative controls must also be incorporated; ideally these should include reagent blanks for all stages of processing and PCR master mix containing probes and primers but no template control (NTC). In practice we include a specimen processed in the same batch as the test samples but known to be negative for the target being analyzed (NAC; no amplification control).

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