DNA and RNA Extractions: Manual vs. (semi-) Automated Procedures

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HistoGeneX Molecular Laboratory (Antwerp, Belgium) is a CAP and CLIA accredited facility and also holds an ISO15189:2012 accreditation (BELAC 257-MED).

We tested different (semi-)automated extraction instruments by extracting molecular material from an equivalent number of adjacent FFPE sections to compare the manual and (semi-)automated procedure. The average extraction yield (orange line, second y-axis, Figures below) was determined using a picogreen and ribogreen assay, respectively. Purity was estimated by the A260/A280 (blue) and A260/A230 (green) ratios. Furthermore, the DNA quality was further assessed by downstream mutation testing, while for RNA, the RIN (RNA Integrity Number) value and DV200 values were calculated using a bio-analyzer.

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A HistoGeneX technologist operating a dissection microscope. The dissection regions are guided by pathology review.

DNA Extractions

While the first generation of automated DNA extraction instruments were rapidly implemented in many global labs, they dramatically underperformed when it came to either yield (automated 2) or purity (automated 1). It appears, however, that instrument performance has improved over the last year. Some automated platforms now equal or even surpass the manual protocols (automated 4 and 5) for both dsDNA yield and purity. Moreover, for some platforms (3 and 5) the ssDNA yield was significantly lower, which may be advantageous when performing capture-based downstream assays instead of PCR approaches. In fact, in e.g. instrument 3a and in the updated version of the instrument 3b, we unequivocally identified PCR-inhibitors which hinder amplification-based downstream tests, thus significantly increasing the rate of invalid samples. It seems that, as automated high-throughput DNA-extraction protocols are optimized and further improved in the near future, they will occupy a spot in the workflow of molecular labs. Clearly, this requires further testing and validation, especially with regards to the performance of the extracted DNA in large panel assays such as tumor mutational burden-assessment.

DNA Extractions DNA HistoBlast
Figure 1. Yield and Purity of DNA Extractions - Manual vs. Automated Systems


RNA Extractions

RNA extraction is even more critical than DNA extraction as the slightest bias or skewing towards shorter or longer fragments may dramatically alter the expression analysis result (e.g., by RNASeq). Over the last 2-3 years we have experienced a shift in volume towards RNA extractions, therefore increasing our interest in automation. There has been a corresponding increase in the number of providers who offer RNA-extraction instruments, protocols and kits. We have evaluated the available automated platforms (1-3, Figure 2) for RNA extraction and intend to evaluate two others that are currently in development. Unfortunately, the (semi-)automated platforms produce significantly lower RNA yields compared to manual procedures. The insufficiency of good quality RNA and the inability to concentrate the RNA through precipitation can reduce assay sensitivity. In turn, this affects the expression profile and presents challenges for low expressing targets. As with its DNA counterpart, RNA (semi-)automated extractions suffer from suboptimal purity (bead-based procedures introduce PCR-inhibitors in the final extract), which may in turn hinder further analysis.

 

RNA Extractions DNA HistoBlast
Figure 2. Yield and Purity of RNA Extractions - Manual versus Automated Systems

Summary

In conclusion, while some instruments that recently entered the market seem very promising, especially where DNA is concerned, it currently appears that automation of the RNA extraction procedure seems to be inextricably linked to large yield losses and a significant reduction in purity. We will continue to extract RNA manually to ensure the optimal yield of good quality RNA for unbiased results in downstream molecular assays. Meanwhile, HistoGeneX will conduct testing and validating automated DNA extractions.

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