To ensure that the results are maximally informative to allow retrospective genetic analyses, we have used a consistent set of historical specimens for all extraction methods, and assessed the quality of the nucleic acids using up to 10 different assays. The specimens themselves are all archival, ranging from 49 to 11 years in age, and have been fixed with either buffered or unbuffered formalin, or with Bouin’s solution. Bouin’s is a picric acid and formaldehyde containing solution that has historically been used predominantly in some European and European colonial laboratories and is notable for its extremely low pH. Different characteristics of extracted nucleic acids may be viewed as important in studies with different aims. Furthermore, because the above may not be linked in a straightforward manner, we have investigated the outcome of the investigated extraction methods using a range of quantitative and qualitative measures. This includes raw quantity of extracted DNA and RNA, PCR amplifiable quantity of human nuclear and mitochondrial DNA, maximum amplifiable length of human nuDNA PCR product, effectiveness in human nuDNA multiplex PCR assays, amplifiable quantity of human RNA following cDNA synthesis through RTPCR, and effectiveness for both proviral DNA, and RT-PCR assays of viral pathogen RNA. Full details are described in the methods section. Because of this range of parameters, and although not a strictly accurate description, we henceforth refer to any increases in any of the chosen measures of nucleic acids as ‘increased quality’of the nucleic acids. Many studies apply pretreatment techniques prior to the extraction of the nucleic acids from the fixed tissues. A number of these are directly related to the removal of the paraffin wax in which many samples are stored.. The most common technique for paraffin removal is based on that described by Goelz et al., using a progression of xylene and ethanol washes. Alternative methods also exist, such as its removal through direct melting using microwaves. The removal of paraffin is believed to be important�Cfor example Stanta et al. argue it otherwise leads to PCR inhibition during subsequent PCR. However, despite this, a growing number of authors take no specific steps to remove the paraffin either due to its substitution with other steps, or simply due to a belief that its removal is unnecessary. The removal of paraffin is not the only pretreatment method that has been described. Formaldehyde induces the cross-linking of protein to other molecules in a manner that is to some extent heat reversible. As such, several pre-digestion heat treatments have been described that confer apparent benefits. These include a 15 minute pre-incubation of the fixed Gomisin-D tissues at 98uC in a conventional Tris-based digestion solution, or, at a greater extreme, preincubation at even higher temperatures in alkali solutions. A further pretreatment that has been reported with specific regard to tissues fixed using Bouin’s solution is washing in 27 mM LiCO3 solution. Although this has not been used in other studies on Bouin’s-fixed samples, and in the original paper the authors do not justify why this is necessary, we presume the aim of the wash is to help remove picric acid from the tissue slices, which may have a detrimental effect, at later stages, on the nucleic acids. From our own observations, we note that the characteristically bright-yellow Bouin’s-fixed tissues rapidly lose their yellow color after one to two brief Ginsenoside-Ro incubations in the LiCO3 solution. However, it remains unclear whether this step is actually warranted to enable the recovery of higher quality nucleic acids.