The Qubit fluorometer is a DNA quantification device based on the fluorescence intensity of fluorescent dye binding to double-stranded DNA (dsDNA). Qubit is generally considered useful for checking DNA quality before next-generation sequencing because it measures intact dsDNA. To examine the most accurate and suitable methods for quantifying DNA for quality assessment, we compared three quantification methods: NanoDrop, which measures UV absorbance; Qubit; and quantitative PCR (qPCR), which measures the abundance of a target gene. For the comparison, we used three types of DNA: 1) DNA extracted from fresh frozen liver tissues (Frozen-DNA); 2) DNA extracted from formalin-fixed, paraffin-embedded liver tissues comparable to those used for Frozen-DNA (FFPE-DNA); and 3) DNA extracted from the remaining fractions after RNA extraction with Trizol reagent (Trizol-DNA). These DNAs were serially diluted with distilled water and measured using three quantification methods. For Frozen-DNA, the Qubit values were not proportional to the dilution ratio, in contrast with the NanoDrop and qPCR values. This non-proportional decrease in Qubit values was dependent on a lower salt concentration, and over 1 mM NaCl in the DNA solution was required for the Qubit measurement. For FFPE-DNA, the Qubit values were proportional to the dilution ratio and were lower than the NanoDrop values. However, electrophoresis revealed that qPCR reflected the degree of DNA fragmentation more accurately than Qubit. Thus, qPCR is superior to Qubit for checking the quality of FFPE-DNA. For Trizol-DNA, the Qubit values were proportional to the dilution ratio and were consistently lower than the NanoDrop values, similar to FFPE-DNA. However, the qPCR values were higher than the NanoDrop values. Electrophoresis with SYBR Green I and single-stranded DNA (ssDNA) quantification demonstrated that Trizol-DNA consisted mostly of non-fragmented ssDNA. Therefore, Qubit is not always the most accurate method for quantifying DNA available for PCR.

Formalin-fixed, paraffin-embedded (FFPE) tissues used for pathological diagnosis are valuable for studying cancer genomics. In particular, laser-capture microdissection of target cells determined by histopathology combined with FFPE tissue section immunohistochemistry (IHC) enables precise analysis by next-generation sequencing (NGS) of the genetic events occurring in cancer. The result is a new strategy for a pathological tool for cancer diagnosis: ‘microgenomics’. To more conveniently and precisely perform microgenomics, we revealed by systematic analysis the following three details regarding FFPE DNA compared with paired frozen tissue DNA. 1) The best quality of FFPE DNA is obtained by tissue fixation with 10% neutral buffered formalin for 1 day and heat treatment of tissue lysates at 95°C for 30 minutes. 2) IHC staining of FFPE tissues decreases the quantity and quality of FFPE DNA to one-fourth, and antigen retrieval (at 120°C for 15 minutes, pH 6.0) is the major reason for this decrease. 3) FFPE DNA prepared as described herein is sufficient for NGS. For non-mutated tissue specimens, no artifactual mutation occurs during FFPE preparation, as shown by precise comparison of NGS of FFPE DNA and paired frozen tissue DNA followed by validation. These results demonstrate that even FFPE tissues used for routine clinical diagnosis can be utilized to obtain reliable NGS data if appropriate conditions of fixation and validation are applied.

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Enantioselective allylations of α-nitro ketones (3) and α-nitro esters (15) with allyl acetate were carried out in the presence of 2 equiv of alkali metal fluorides (KF, RbF, CsF) and 1 mol % of palladium catalysts prepared in situ from Pd2(dba)3·CHCl3 and chiral phosphine ligands. Moderate enantioselectivities were observed in the reaction of nitro ketones 3, giving products 4 (4a, 49% ee; 4b, 58% ee; 4c, 44% ee) when rubidium fluoride and ferrocenylphosphine ligands bearing monoaza-15-crown-5 (1b) or monoaza-18-crown-6 (1c) moieties were used as a base and a chiral ligand, respectively. Optically active allylation product 4a was converted into 1-methyl-1-azaspiro[4.5]decan-10-amine (13), a precursor to opioid receptor binding agents. Enantioselectivity in the reaction of nitro esters 15 increased in accord with increasing steric demand of the ester alkyl group (Me < Et < t-Bu). The highest selectivity (80% ee) for the reaction of tert-butyl ester 15c was observed when the reaction was carried out at −40 °C in the presence of the palladium catalyst with the ligand (1c) bearing a monoaza-18-crown-6 moiety, RbF (2 equiv), and RbClO4 (1 equiv). The pronounced effect of the crown ether moiety for both enantioselection and rate acceleration can be explained by assuming the formation of a ternary complex involving the crown ether, rubidium cation, and enolate anion at the stereodifferentiating transition state. Optically active nitro ester (R)-16c was converted into (R)-α-methylglutamic acid (20).