Circulating nucleic acids (CNAs) are under investigation as a liquid biopsy in cancer. However there is wide variation in blood processing and methods for isolation of circulating free DNA (cfDNA) and microRNAs (miRNAs). Here we compare the extraction efficiency and reproducibility of 4 commercially available kits for cfDNA and 3 for miRNA using spike-in of reference templates. We also compare the effects of increasing time between venepuncture and centrifugation and differential centrifugation force on recovery of CNAs. cfDNA was quantified by TaqMan qPCR and targeted deep sequencing. miRNA profiles were assessed with TaqMan low-density arrays and assays. The QIAamp® DNA Blood Mini and Circulating nucleic acid kits gave the highest recovery of cfDNA and efficient recovery (>90%) of a 564bp spike-in. Moreover, targeted sequencing revealed overlapping cfDNA profiles and variant depth, including detection of HER2 gene amplification, using the Ion AmpliSeq™Cancer Hotspot Panel v2. Highest yields of miRNA and the synthetic Arabidopsis thaliana miR-159a spike-in were obtained using the miRNeasy Serum/Plasma kit, with saturation above 200 µl of plasma. miRNA profiles showed significant variation with increasing time before centrifugation (p<0.001) and increasing centrifugation force, with depletion of platelet associated miRNAs, whereas cfDNA was unaffected. However, sample replicates showed excellent reproducibility on TaqMan low density arrays (ρ = 0.96, p<0.0001). We also successfully generated miRNA profiles for plasma samples stored > 12 years, highlighting the potential for analysis of stored sample biobanks. In the era of the liquid biopsy, standardisation of methods is required to minimise variation, particularly for miRNA.

DNA extracted from forensic samples can be degraded and also contain co-extracted contaminants that inhibit PCR. The effects of DNA degradation and PCR inhibition are often indistinguishable when examining a DNA profile. Two internal amplification controls (IACs) were developed to improve quality control of PCR using the AmpFℓSTR® SGM Plus® kit. The co-amplification of these controls with DNA samples was used to monitor amplification efficiency and detect PCR inhibitors. IAC fragments of 90 and 410 bp (IAC₉₀ and IAC₄₁₀) were generated from the plasmid pBR322 using tailed primers and then amplified with ROX-labelled primers. Co-amplification of IAC₉₀ and IAC₄₁₀ was performed with varying amounts of template DNA, degraded DNA and DNA contaminated with humic acid, heme and indigo dye. Both IAC₉₀ and IAC₄₁₀ were successfully amplified with human DNA without significantly affecting the quality of the DNA profile, even with DNA amounts lower than 0.5 ng. In the presence of inhibitors, the IAC₉₀ signal was still present after all human DNA loci fail to amplify; in contrast, the IAC₄₁₀ signal was reduced or absent at low levels of inhibition. Amplification of the two IACs provided an internal PCR control and allowed partial profiles caused by inhibition to be distinguished from degraded DNA profiles.

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An investigation into the effects of physical and chemical enhancement on subsequent presumptive and confirmatory tests for human blood is presented. Human blood was deposited onto porous (white 80gsm paper and brown envelope) and non-porous (tile and linoleum) substrates in a depletion series (30 depletions on non-porous and 20 on porous) and subjected to three ageing periods; 1, 7 and 28 days. A number of enhancement techniques were tested [fluorescence, black magnetic powder (BMP), iron-oxide black powder suspension (PS), cyanoacrylate (CA) fuming, acid violet 17 (AV17), acid yellow 7 (AY7), ninhydrin, DFO and Bluestar Forensic Magnum (BFM) luminol] to evaluate their potential effects on subsequent presumptive and confirmatory tests. AV17 and Bluestar provided the best enhancement and fully enhanced all depletions in the series. The sensitivity of the Kastle-Meyer (KM) (presumptive), Takayama and RSID-Blood tests (confirmatory) was initially investigated to determine the range of detectable depletions. The KM test detected all depletions, whereas the Takayama test detected up to depletion 6 and RSID-Blood detected up to depletion 20 (paper), 10 (envelope), 15 (tile) and 9 (lino). The abilities of these tests to detect blood after enhancement were then observed.A number of techniques resulted in little to no effect on any of the blood tests, whereas adverse effects were observed for others. Ninhydrin and CA fuming caused weak but instantaneous positive KM results whereas methanol-based AV17 and AY7 delayed the reaction by as much as 1 minute. The Takayama test was not very sensitive, therefore, its performance was easily affected by enhancement and negative results were often observed. RSID-Blood tests were largely unaffected by chemical enhancement although a drop in positive results was observed for some of the techniques when compared to positive controls.Using a standard procedure for DNA extraction, all the tested blood samples (before and after enhancement) gave a detectable quantity of DNA and were successfully profiled. Out of the 45 samples processed for DNA profiling, 44 gave full profiles, while the remaining showed allele drop out in one or two loci.

In this study, we have developed a PCR multiplex that can be used to assess DNA degradation and at the same time monitor for inhibition: primers have been designed to amplify human, pig, and rabbit DNA, allowing pig and rabbit to be used as experimental models for taphonomic research, but also enabling studies on human DNA persistence in forensic evidence. Internal amplified controls have been added to monitor for inhibition, allowing the effects of degradation and inhibition to be differentiated. Sequence data for single-copy nuclear recombination activation gene (RAG-1) from human, pig, and rabbit were aligned to identify conserved regions and primers were designed that targeted amplicons of 70, 194, 305, and 384 bp. Robust amplification in all three species was possible using as little as 0.3 ng of template DNA. These have been combined with primers that will amplify a bacterial DNA template within the PCR. The multiplex has been evaluated in a series of experiments to gain more knowledge of DNA persistence in soft tissues, which can be important when assessing what material to collect following events such as mass disasters or conflict, when muscle or bone material can be used to aid with the identification of human remains. The experiments used pigs as a model species. When whole pig bodies were exposed to the environment in Northwest England, DNA in muscle tissue persisted for over 24 days in the summer and over 77 days in the winter, with full profiles generated from these samples. In addition to time, accumulated degree days (ADD) were also used as a measure that combines both time and temperature-24 days was in summer equivalent to 295 ADD whereas 77 days in winter was equivalent to 494 ADD.

Biological samples recovered for forensic investigations are often degraded and/or have low amounts of DNA; in addition, in some instances the samples may be contaminated with chemicals that can act as PCR inhibitors. As a consequence this can make interpretation of the results challenging with the possibility of having partial profiles and false negative results. Because of the impact of DNA analysis on forensic investigations, it is important to monitor the process of DNA profiling, in particular the amplification reaction. In this chapter we describe a method for the in-house generation and use of internal amplification controls (IACs) with DNA profiling kits to monitor the success of the PCR proces. In the example we show the use of the SGM Plus® kit. These controls can also be used to aid the interpretation of the DNA profile.

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