Sample prep for AD research: FFPE tissue
The fixation of brain tissue with formaldehyde (also known as formalin), followed by tissue dehydration and embedding into a paraffin block, is a common method of preservation that has been used for almost a century. This approach renders tissue remarkably stable, allowing the long-term storage (years to decades) of Alzheimer's and control tissue in ambient conditions. These FFPE blocks are highly compatible with immunohistochemical analyses of protein expression and cell morphology and, as a result, research and clinical institutions have accumulated enormous biobanks of human tissue that may consist of thousands of samples. These samples can span several decades and are linked with relevant genetic, clinical, and diagnostic data. Of particular utility for CNS researchers is that tissue blocks may be sorted by neuroanatomical region, a key consideration in understanding the progression and impact of Alzheimer's disease.
While FFPE tissue provides an incredibly valuable research tool, the fixation process causes extensive nucleic acid/protein crosslinking, DNA and RNA fragmentation, and the deamination and/or oxidation of bases, rendering FFPE samples a challenging source of DNA and RNA. Fortunately, specialized methods have been developed for nucleic acid extraction from FFPE samples that yield outputs suitable for applications such as PCR and next-generation sequencing.
Please note that Macherey-Nagel nucleic acid purification products are only available from Takara Bio in North America, India, and Japan.
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DNA purification
Takara Bio offers both silica membrane- and magnetic bead-based technologies for the purification of DNA from a wide array of FFPE tissue types with the NucleoSpin DNA FFPE XS and NucleoMag DNA FFPE kits. Both technologies incorporate the use of an odorless, non-toxic, xylene-free reagent for paraffin removal. Samples are then treated with proteinase to solubilize the fixed tissue and release DNA into solution, followed by heat incubation in a specially formulated buffer to eliminate crosslinks. Lysate DNA is then bound to a silica membrane or paramagnetic beads, subjected to sequential wash steps, and eluted. The NucleoSpin FFPE DNA kit is provided in both single- and multi-prep formats for varying throughput demands, whereas the NucleoMag FFPE DNA kit has been developed primarily for automated, high-throughput processing.
Figure 1. Outstanding yield and PCR performance of de-crosslinked DNA with NucleoSpin DNA FFPE kits. Panel A. DNA was isolated with NucleoMag DNA FFPE from 1 slice of mouse FFPE tissue section/prep (4 preps for each tissue type). DNA yield (dark blue bars) was quantified via optical density, and qPCR analyses (orange squares) were performed using the Thermo Fisher Scientific Maxima SYBR Green qPCR Master Mix. Panel B. DNA was isolated from FFPE rat liver tissue with NucleoSpin DNA FFPE XS (2x blue graphs) and with an FFPE mini elution kit from Competitor Q (2x orange graphs), and a 100-bp target was amplified by PCR using a Roche LightCycler system. Yields were consistently higher with NucleoSpin DNA FFPE XS, and the DNA provided better PCR performance relative to the output from Competitor Q's kit. For this comparison, the starting sample was one section of FFPE tissue that was subjected to overnight lysis and eluted in a volume of 30 µl.
RNA purification
For purification of total RNA from FFPE samples, Takara Bio offers silica membrane-based technologies in single-prep formats designed for varying input amounts: NucleoSpin totalRNA FFPE and NucleoSpin totalRNA FFPE XS. Following deparaffinization, proteinase treatment, and heating, the lysate is applied to the membrane under conditions that enable binding of diverse RNA species, including small RNAs. Residual DNA is then digested via an on-column DNase treatment and, following successive washing steps, RNA is eluted in a small volume of RNase-free water, yielding highly concentrated RNA with superior yield and lower gDNA contamination than competitors' kits (Figure 2).
Figure 2. NucleoSpin totalRNA FFPE provides higher RNA yields and lower DNA contamination from brain tissue relative to competitor's kits. Total RNA was isolated from four 10-µm thick FFPE sections of mouse brain tissue with NucleoSpin totalRNA FFPE (MN) and three competitor kits (Q, A, and P). Panel A. mRNA and miRNA targets were amplified by qPCR (the mRNA target consisted of a 230-nt portion of the β2-microglobulin gene, while the miRNA target was analyzed using the miR-16 TaqMan MicroRNA Assay. (Note: miRNA samples were reverse transcribed separately using the TaqMan MicroRNA Reverse Transcription Kit from Thermo Fisher Scientific.) Panel B. Residual DNA was assayed by amplifying a 191-bp fragment of the mGAPDH gene. For both assays, the lower CT values observed for samples processed with the NucleoSpin totalRNA FFPE kit provided higher RNA yields and lower DNA contamination relative to competitor's kits.
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