With the emerging applications of Next Generation Sequencing (NGS) technologies there is a need for fast and reliable nucleic acid extraction methods. In a clinical setting the available samples pose challenges to downstream applications. Often only small amount of biopsy samples are available and their nucleic acid content can be altered due to chemical processing. To accurately profile mutations using NGS it is necessary to maximise good quality DNA or RNA output from the smallest amount of samples. With the increasing throughput of sample processing in a molecular pathology lab the compatibility of the available methods with lab automation is desired.
Several products are available in the Thermo Fisher Scientific portfolio for nucleic acid isolation depending on your sample type and downstream application. With these kits a 2 day workflow from blood to NGS data became feasible. See table below for an overview of nucleic acid Isolation kits for various sample types.
The two main sample types available for clinical molecular testing are FFPE and cfDNA samples. ThermoFischer Scientific provides complete solutions for processing these challenging sample types from sample to qPCR, digital PCR, Sanger sequencing or NGS data.
FFPE tissue samples
Formalin-fixed, paraffin embedded (FFPE) tissues are often used for histopathological analysis. Fixed samples also have the advantage of long term storage for retrospective research or complete historical analysis with clinical information on treatments and outcomes. Due to the fixation procedure and chemicals used the nucleic acid material (DNA, RNA) can go through chemical changes, cross-linking and degradation. The recovered DNA is not optimal for whole genome sequencing due to this fragmentation process, but still provides excellent material for targeted gene studies such as qPCR, dPCR, Sanger sequencing and Targeted NGS. The goal during sample preparation for NGS or dPCR is to maximise target presence in order to achieve reliable variant detection. This can be achieved by macro-and microdissection of the relevant tissue section utilising expertise in Histopathology. It is critical to maximise nucleic acid extraction from a small amount of tissue sample. The LOD (level of detection) value is different depending on the sequencing panel used, up-stream sample treatment and storage, or the nucleic acid isolation technique applied.
The IonAmpliseq Direct FFPE kit provides a two-step, single tube DNA extraction method. It eliminates the need for harmful de-paraffinisation chemicals such as xylene. The method was originally developed for IonAmpliseq library preparation to accommodate 1-100ng DNA input range. The two pipetting steps with 10 min. hands-on time only takes 30 minutes to perform. An optional Uracil DNA Glycosylase (UDG) protocol is available to remove deaminated cytosines for more reliable variant analysis. The resulting DNA sample is suitable for not only NGS library preparation and sequencing but also for Sanger sequencing, quantitative or digital PCR applications and rare mutation detection.
The IonTorrent Oncomine™ panels (Oncomine™ Focus Assay, Oncomine™ Comprehensive Assay) benefit from both DNA and RNA target sequencing and analysis. The combined gene expression, fusion event and DNA sequence analysis data is then used for variant calling and interpretation of carried mutations in the cancerous cells. It is desirable that the same tissue section is processed after microdissection for both DNA and RNA analysis. Thermo Fisher Scientific offers two products for ‘same-sample’ RNA DNA extraction.
For FFPE gene expression studies and sequencing the RecoverAll™ Total Nucleic Acid Isolation kit offers sequential RNA and DNA isolation from FFPE samples. The total RNA content of embedded cells are heavily degraded and different RNA extraction kits can have a bias in recovering larger or smaller fragment sizes thus the gene expression studies can be compromised. The Recover All™ kit is designed to release the maximal amount of RNA fragments of all sizes including miRNA from de-paraffinized tissue material. The kit can also be used with limited amount of samples, needle biopsies, laser captured microdissections tissues for both DNA and RNA isolation.
A magnetic based alternative for sequential RNA and DNA isolation is the MagMAX FFPE DNA/RNA Ultra Kit. The kit demonstrated excellent performance for both xylene based and CitriSolv based de-paraffinised samples. The kit is sufficient for nucleic acid extraction from up to 40 µm sections, however by adding the Supplemental Reagents kit, larger than 40 µm sections can be extracted and the yield increased. The protocol can be used manually or automated with KingFisher Flex™ Magnetic Particle Processor and KingFisher™ Duo Magnetic Bead Processor. Lab automation is minimising hands-on sample extraction time and possible contamination and helps increasing sample processing throughput in a busy clinical laboratory.
Liquid biopsy and cell-free DNA
In precision medicine drugs are developed and used for targeting driver genes involved tumour progression. Before treatment with targeted therapies it is essential to confirm mutation using companion diagnostics. Liquid biopsy became an indispensable tool for monitoring cancer patients and diagnosing drug tolerance over treatment period. Using cfDNA from peripheral blood is of interest to many researchers studying practical methods to replace a tissue biopsy. Advantages of liquid biopsy:
Multiple sample, serial analysis of sample
Better analysis of tumour heterogeneity
Hard to collect tissue samples ie. lung
Less invasive less expensive than tissue
Often difficult to conduct second biopsy after treatment
Digital PCR, Sanger Sequencing and NGS are sensitive methods to detect tumour-derived mutations at low frequency and they are compatible with a small amount of nucleic acid. The IonTorrent™ Oncomine™ cfDNA assays detect single nucleotide variants, insertions or deletions at 0.1-1% typical allele frequency in circulating tumour DNA (ctDNA). There are three cfDNA panels; the Oncomine™ Lung cfDNA Assay, the Oncomine™ Colon cfDNA Assay and the Oncomine™ Breast cfDNA Assay available from IonTorrent™ but researchers also analysed and described abnormalities using cfDNA from lung, breast, pancreatic, colon, and prostate cancer. 10mL blood has 5-20 ng cfDNA in healthy patients and 5-100 ng at late stage cancer. The MagMAX cell-free DNA Isolation Kit is using Dynabeads™ MyOne™ SILANE technology and is optimised for reproducible recovery of cell-free DNA with minimal background of large genomic DNA. The magnetic bead-based extraction method is adaptable to automation. There is no need for Proteinase and heat treatment further simplifying the extraction workflow. High quality of cfDNA can be isolated within 40 minutes at room temperature. The sample processing is scalable and well suited for higher throughput laboratories.
|Kit Name||Input type||Input size||Output||Price per Sample|
|GeneCatcher gDNA||Blood||0.3-1 mL||_||5-23 €|
|Blood||10 mL||300 µg|
|iPrep™ PureLink® gDNA Blood Kit||Blood||0.35 mL||15 µg||6.5€|
|TRIzol® Reagent||Cells||5x106 cells||_||3-3.5 €|
|DNAzol® Reagent||Blood||0.1 mL||_||2.7 €|
|DNAzol® BD Reagent||Blood||0.5 mL||10-20 µg||2.7 €|
|PureLink Genomic DNA Mini (filter column)||Blood||0.2 mL||3-10 µg||2.6-3.8 €|
|Buccal swabs||1 swab||6 µg|
|Cells||5x106 cells||10 µg|
|Tissue||25 mg||5-10 µg|
|MagMAX™ DNA Multi-Sample Kits / MagMAX™ DNA Multi-Sample 96-Well Kits||Blood||50-350 µL||1.5-16 µg||2.5-3.2 €|
|Buccal swabs||1 swab||2-12 µg|
|Cells||5x106 cells||15-30 µg|
|Tissue||25-50 mg||10-80 µg|
|Ion AmpliSeq™ Direct FFPE DNA Kit||FFPE tissue||5-10µm section, 4-100 mm2 tissue||DNA only||6.3-7.5 €|
|RecoverAll™ Total Nucleic Acid Isolation Kit||FFPE tissue||4x20µm section, 300 mm2 tissue||20x50µL DNA and 20x50µL RNA||3.8-10.9 €|
|60x50µL DNA and 60x50µL RNA|
|100x50µL DNA and 100x50µL RNA|
|MagMAX™ FFPE DNA/RNA Ultra Kit||FFPE tissue||2x20µm section, 300 mm2 tissue||48x50µL DNA and 48x50µL RNA||5.2-6.7 €|
|Supplemental reagents for use with the MagMAX FFPE DNA/RNA Ultra Kit||FFPE tissue||>40µm section tissue||NA|
|MagMAX™ Cell-Free DNA Isolation Kit||plasma, serum, urine||05-10 mL||cf DNA||9.5 €|
|Ambion®RiboPure™-Blood Kit||blood||0.5 mL||2-4 µg||16.5 €|
|Ambion® RiboPure™ Kit||cells||2x107 cells||100-500 µg||11.60 €|
|PureLink® Total RNA Blood Kit||blood||50-500 µL||up to 1000 µg||6.3 €|
|iPrep™ PureLink® Total RNA Kit||Blood||1 mL||_||7.5 €|
|MagMAX™ for Stabilized Blood Tubes RNA Isolation Kit (Tempus Blood Tubes)||Blood||3 mL||3-25 µg||11.0 €|
|MagMAX™ for Stabilized Blood Tubes RNA Isolation Kit (PaxGene Blood Tubes)||Blood||5 mL||3-18 µg||11.0 €|
|MagMAX™-96 Blood RNA Isolation Kit||Blood||50 µL||300-500 ng||6.3 €|
|PureLink® RNA Mini Kit||blood||0.2 mL||up to 1000 µg||5.3-13.2 €|
|PureLink® RNA Micro Scale Kit||cells||105 cells||100 µg||7.2 €|
|PureLink® FFPE RNA Isolation Kit||FFPE tissue||3-8x10µm sections||up to 1 µg||6.4 €|
|MagMAX™ mirVana™ Total RNA Isolation Kit||cells||5x106 cells||_||5.1 €|
|plasma, serum||100 µL|
|PureLink® Pro 96 Viral RNA⁄DNA Purification Kit||plasma, serum, CSF||0.2 mL||_||2.9 €|
|PureLink® Viral RNA/DNA Mini Kit||plasma, serum, CSF||0.5 mL||_||4.1 €|
|MagMAX™ Pathogen RNA/DNA Kit||blod, plasma, semen, feces, InPouch cultures swabs etc. with low abundance pathogen||50-350 µL||_||3.3 €|
Please see ordering information below.
|Kit Name||Product code||Product size/throughput||Price||Price per sample|
|GeneCatcher gDNA||CS21101||96||€481.00||€ 5.01|
|iPrep™ PureLink® gDNA Blood Kit||IS10005||52||€340.00||€ 6.54|
|TRIzol® Reagent||10296010||100||€344.00||€ 3.44|
|DNAzol® Reagent||10503027||100||€266.00||€ 2.66|
|DNAzol® BD Reagent||10974020||100||€272.00||€ 2.72|
|PureLink Genomic DNA Mini||K182000||10||€38.32||€ 3.83|
|MagMAX™ DNA Multi-Sample Kits||4413020||50||€160.00||€ 3.20|
|MagMAX™ DNA Multi-Sample 96-Well Kits||4413021||96||€297.00||€ 3.09|
|MagMAX™ DNA Multi-Sample Ultra Kit||A25597||500||€1,414.00||€ 2.83|
|Ion AmpliSeq™ Direct FFPE DNA Kit||A31133||8||€60.50||€ 7.56|
|RecoverAll™ Total Nucleic Acid Isolation Kit||AM1975||40||€434.00||€ 10.85|
|RecoverAll™ Multi-Sample RNA/DNA Workflow||A26069||120||€476.00||€ 3.97|
|MagMAX™ FFPE DNA/RNA Ultra Kit||A31881||96||€495.00||€ 5.16|
|Supplemental reagents for use with the MagMAX FFPE DNA/RNA Ultra Kit||A32796||96||€144.00||€ 1.50|
|MagMAX™ Cell-Free DNA Isolation Kit||A29319||50||€474.00||€ 9.48|
|RiboPure™-Blood Kit||AM1928||40||€658.00||€ 16.45|
|Ambion® RiboPure™ Kit||AM1924||50||€578.00||€ 11.56|
|PureLink® Total RNA Blood Kit||K156001||50||€315.00||€ 6.30|
|iPrep™ PureLink® gDNA Blood Kit||IS10006||52||€390.00||€ 7.50|
|MagMAX™ for Stabilized Blood Tubes RNA Isolation Kit (Tempus Blood Tubes)||4451893||96||€1,062.00||€ 11.06|
|MagMAX™ for Stabilized Blood Tubes RNA Isolation Kit (PaxGene Blood Tubes)||4451894||96||€1,050.00||€ 10.94|
|MagMAX™-96 Blood RNA Isolation Kit||AM1837||96||€602.00||€ 6.27|
|PureLink® RNA Mini Kit||12183020||10||€132.00||€ 13.20|
|PureLink® RNA Micro Scale Kit||12183016||50||€360.00||€ 7.20|
|PureLink® FFPE RNA Isolation Kit||K156002||50||€318.00||€ 6.36|
|MagMAX™ mirVana™ Total RNA Isolation Kit||A27828||96||€485.00||€ 5.05|
|PureLink® Pro 96 Viral RNA⁄DNA Purification Kit||12280096A||384||€1,096.00||€ 2.85|
|PureLink® Viral RNA/DNA Mini Kit||12280050||50||€207.00||€ 4.14|
|MagMAX™ Pathogen RNA/DNA Kit||4462359||480||€1,602.00||€ 3.34|
The Thermo Scientific™ KingFisher™ purification systems are designed to deliver high quality results with minimal hands-on time. These systems can be a useful addition to laboratories not only processing samples for downstream nucleic acid analysis but there are suitable kits available for pathogen RNA and DNA isolation, mRNA isolation or Proteomics studies.
KingFisher™ Flex system enables processing 24 or 96 samples per run. Suitable for nucleic acid extraction from various starting materials combined with MagMAX FFPE DNA/RNA Ultra Kit or MagMAX cell-free DNA Isolation Kit. For smaller sample throughput labs the KingFisher™ Duo Prime system is recommended with slightly smaller footprint and 12-24 sample processing..
For downstream processing of purified nucleic acid samples the use of Qubit™ 3.0 or its previous generation is strongly recommended. Based on the detection of target-specific fluorescence, this integrated system is more sensitive than UV absorbance–based quantification, making it ideal for precious samples and demanding applications.
Gyongyi Lukacs, PhD
Field Application Specialist