Improving Yields in Multi-analyte Extractions by Utilizing Post-homogenized Tissue Debris

Abstract

In multi-analyte extractions, tissue is typically homogenized in a lysis buffer, and then DNA, RNA, and protein are purified from the supernatant. However, yields are typically lower than in dedicated, single-analyte extractions. In a two-part experiment, we assessed whether yields could be improved by revisiting the normally discarded, post-homogenized tissue debris. We initially performed additional homogenizations, each followed by a simultaneous extraction. These yielded no additional RNA, 13% additional DNA (which became progressively more degraded), and 161.7% additional protein (which changed in proteome when analyzed using SDS-PAGE). We then digested post-homogenized tissue debris from a simultaneous extraction using proteinase K and extracted DNA using silica spin columns or alcohol precipitation. An average additional DNA yield of 27.1% (silica spin columns) or 203.9% (alcohol precipitation) was obtained with/without compromising DNA integrity (assessment by long-range PCR, DNA Integrity Numbers, and size at peak fluorescence of electropherogram). Validation using a cohort of 65 tissue blocks returned an average additional DNA yield of 31.6% (silica columns) and 54.8% (alcohol precipitation). Users can therefore refreeze the homogenized remnants of tissue blocks rather than disposing of them and then perform additional DNA extractions if yields in the initial multi-analyte extractions were low.

Keywords: AllPrep; Puregene; QIAamp; biospecimen science; homogenization; optimization; simultaneous.

Figure 1.

DNA yield from tissue blocks that underwent four sequential homogenizations (Hom 1–4), each followed by an AllPrep simultaneous DNA/RNA/protein extraction.

Figure 2.

Yield and consistency in protein extracted from tissue blocks after four sequential homogenizations (Hom 1–4), each followed by a simultaneous DNA/RNA/protein extraction. (A) Protein yield (n=11). (B) Commonality in protein bands detected following SDS-PAGE when tissue blocks (n=6) underwent three sequential homogenizations (Hom 2–4). (C) The magnitude of change in volume (either increasing or decreasing) of the protein bands of Fig. 2B following Hom 3 and 4 compared with Hom 2. Data are mean number of bands per tissue block. (D) Gel image of the banding pattern of a tissue block following Hom 2, 3, and 4. The black arrow denotes a band detected in Hom 2 and 3 only, the gray arrow a band with volume increases of 13% between Hom 2 and 3 and then 15% between Hom 2 and 4, and the white arrow a band with a volume decrease of 34% between Hom 2 and 3 and then a volume increase of 28% between Hom 2 and 4.

Figure 3.

DNA yield from tissue blocks that were homogenized for AllPrep simultaneous DNA/RNA/protein extractions, followed by DNA-only extractions from the tissue block debris. The DNA-only extractions were performed using either the QIAamp kit (A) or the Puregene kit (B), after the tissue debris had undergone a proteinase K digest.

Figure 4.

Purity and integrity of DNA extracted from homogenized tissue blocks using the AllPrep simultaneous DNA/RNA/protein extraction kit compared with that extracted from the post-homogenized tissue debris using either the QIAamp or Puregene DNA-only kits. (A) 260:280 purity ratio, (B) DIN, and (C) % dsDNA. Data from the two cohorts of AllPrep extractions were very similar, so were pooled for the purposes of brevity in the Figure, but analyses were performed using non-pooled data. The boxes extend through the second and third quartiles, intersected with the median, and the whiskers extend to the 10th and 90th percentile and the dots are the remaining data points. * denotes statistical significance. (D) Overlaid electropherograms from TapeStation DIN analysis for two representative samples: the peak at 100 bp is the lower marker, the center peak is a DNA sample of 233 ng/µl with DIN 6.2 and maximum fluorescence assigned at 4640 bp, and the right peak is a DNA sample of 152 ng/µl with DIN of 9.1 and maximum fluorescence assigned at >60,000 bp. Abbreviation: DIN, DNA Integrity Number; dsDNA, double-stranded DNA.

Figure 5.

DNA yield and integrity in the validation cohort. (A) DNA yield from AllPrep extractions from homogenized tissue blocks, followed by either QIAamp (n=33) or Puregene (n=32) extractions from proteinase K–digested, residual tissue debris. Data are presented in ascending order of DNA yield in the initial AllPrep extraction. (B) DINs in the QIAamp and Puregene extractions. (C) Size (in kbp) at the point in the electropherograms at peak fluorescence (i.e., at the greatest abundance of DNA). The parameters of the box plots are as in Fig. 4. The differences between QIAamp and Puregene were not statistically significant in either B or C. Abbreviation: DIN, DNA Integrity Number.