Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Reliability and performance of commercial RNA and DNA extraction kits for FFPE tissue cores.

Author(s): Patel PG, Selvarajah S, Guérard KP, Bartlett JMS, Lapointe J, Berman DM, Okello JBA, Park PC

Publication: PLoS One, 2017, Vol. 12, Page e0179732

PubMed ID: 28640876 PubMed Review Paper? No

Purpose of Paper

This paper compared the effects of extraction kit on nucleic acid yield, purity, integrity, and NanoString and methylation-specific PCR results in pooled FFPE prostate specimens. The effects of specimen storage duration on nucleic acid yield and quality and inter-laboratory variability were also investigated.

Conclusion of Paper

RNA purity as assessed by ratio of absorbance at 260/280 (A260/A280) was high regardless of extraction kit but only extraction with the RNeasy FFPE (RNeasy) kit resulted in a A260/A230 close to 2 and yields were highest when extraction was with the PureLink FFPE RNA Isolation (Purelink) Kit. The percentage of fragments >200 bp (DV200) was highest when RNA was extracted using Norgen FFPE RNA Purification (Norgen) or High Pure FFPET RNA Isolation (HighPure) kits. Although amplification of β-2-M fragments ≤303 bp was possible in RNA extracted with the four kits tested, the 386 bp product was only amplifiable in RNA extracted with the RecoverAll Total Nucleic Acid Isolation Kit for FFPE (RecoverAll) or the AllPrep DNA/RNA FFPE (AllPrep) kits. There were no significant differences in NanoString signal counts between methods. The real-time RT-PCR quantification cycle (Cq) values for all three transcripts were higher when extracted with AllPrep compared to other kits (P<0.0001), but the authors state this effect would likely be eliminated by dilution. For specimens extracted using AllPrep, there was no effect of duration of FFPE block storage on the RNA yield, purity, or NanoString counts and results were comparable among the three laboratories performing the extractions.

The highest DNA yields were obtained after extraction with the DNeasy Blood & Tissue (DNeasy) kit. Similar to the RNA results, the A260/A280 ratios were generally high but half of the kits had low A260/A230 ratios, indicating the presence of organic contaminants. Interestingly, only the contaminants present after extraction with AllPrep had an inhibitory effect on the amplification of a mouse gene and this effect was eliminated when DNA was diluted 10-fold. Each of the five kits prioritized for further testing allowed for amplification of all four PCR products (102-300 bp), but the Cq values for the three methylation-specific real-time PCR were lowest when DNA was extracted with the AllPrep, DNeasy, or GeneJET Genomic DNA Purification (GeneJet) kits.

Studies

Study Purpose

The purpose of this study was to compare the yield, purity and integrity of RNA obtained from FFPE specimens using eight different extraction kits and to evaluate the suitability of the RNA extracted using four different kits for NanoString and real-time RT-PCR. Tumor-rich areas in four FFPE archival prostate specimens were cored (0.6 mm) and the resultant 120 cores were pooled, deparaffinized in two changes of xylene, washed, and suspended in ethanol. Specimens were then homogenized and aliquoted for extraction using each of the eight kits. Nucleic acids were extracted following the manufacturer’s instructions with the RecoverAll Total Nucleic Acid Isolation Kit for FFPE, AllPrep DNA/RNA FFPE Kit, PureLink FFPE RNA Isolation Kit, E.Z.N.A. FFPE RNA Kit, RNeasy FFPE Kit, High Pure FFPET RNA Isolation Kit, Norgen FFPE RNA Purification Kit, and NucleoSpin total RNA FFPE XS kits. RNA yields were determined using the RNA BR (Broad-Range) Assay kit, purity was determined by spectrophotometric absorbance ratios, and integrity was measured by Bioanalyzer DV200. The presence of inhibitory substances was examined by real-time PCR amplification of the mouse HSD11β1 gene in cell-line DNA spiked with the extracted RNA. The size distribution of RNA was determined by amplification of 92, 142, 200, 248, 303, and 386 bp fragments of β-2-microglobulin. The suitability for expression analysis of RNA extracted using the RecoverAll, ALLPrep, PureLink and RNeasy kits was investigated by hybridization to NanoString 48-plex Customer Assay Evaluation and by TaqMan real-time PCR amplification of three housekeeping genes: Phosphoglycerate Kinase 1 (PGK1), Keratin 8 (KRT8), and Hypoxanthine Phosphoribosyltransferase 1 (HPRT1).

Summary of Findings:

RNA yields were highest when extraction was performed with the PureLink kit (3603.48 ng/mm³) followed by the RNeasy (2713.04 ng/mm³), AllPrep (2512.08 ng/mm³), HighPure RNA (2288.89 ng/mm³), and RecoverAll (2249.95 ng/mm³) kits. Lower yields were obtained using the Norgen RNA (1662.61 ng/mm³), EZNA (1845.12 ng/mm³), and NucleoSpin RNA (985.99 ng/mm³) kits. While the purity of the RNA as assessed by ratio of absorbance at 260/280 (A260/A280) was high (1.85-2.01) regardless of extraction method, only RNA extracted with the RNeasy kit had a A260/A230 close to 2 (2.11). However, the impurities did not have an inhibitory effect on PCR as the real-time PCR cycle threshold values for amplification of a mouse gene did not change with the addition of extracted RNA, regardless of extraction method. The percentage of fragments >200 bp (DV200) was highest when RNA was extracted using the Norgen RNA kit (70%) or HighPure RNA (69%) followed by PureLink (62%), AllPrep (54%), Recoverall (43%), Rneasy (41%), EZNA RNA (31%), and NucleoSpin RNA (10%) kits.

Amplification of β-2-M fragments ≤303 bp was possible in RNA extracted with the four kits prioritized for further testing (AllPrep, PureLink, RNeasy, RecoverAll) but the 386 bp product was only amplifiable in RNA extracted with the RecoverAll and AllPrep kits. NanoString was successful using RNA extracted with each of the four prioritized kits and there were no significant differences in total signal counts, but the relative intensity of the signals varied between methods. Similarly, real-time RT-PCR amplification of all three housekeeping genes was successful using RNA extracted with each of the four kits, but as expected based on the PCR inhibition results, the quantification cycle (Cq) values for all three were higher when RNA was extracted with the AllPrep kit compared to other kits (P<0.0001).

Biospecimens

Tissue - Prostate

Preservative Types

Formalin

Diagnoses:

Neoplastic - Carcinoma

Platform:

Analyte	Technology Platform
RNA	Fluorometry
RNA	Real-time qRT-PCR
RNA	DNA microarray
RNA	Spectrophotometry

Pre-analytical Factors:

Classification	Pre-analytical Factor	Value(s)
Analyte Extraction and Purification	Analyte isolation method	RecoverAll Total Nucleic Acid Isolation Kit for FFPE AllPrep DNA/RNA FFPE Kit PureLink FFPE RNA Isolation Kit E.Z.N.A. FFPE RNA Kit RNeasy FFPE Kit High Pure FFPET RNA Isolation Kit Norgen FFPE RNA Purification Kit NucleoSpin total RNA FFPE XS kits
Real-time qRT-PCR Specific	Targeted nucleic acid	B2M HPRT PGK1 KRT8
Real-time qRT-PCR Specific	Length of gene fragment	92 bp 142 bp 200 bp 248 bp 303 bp 386 bp

Study Purpose

The purpose of this study was to compare the yield, purity, and amplifiability of DNA obtained from FFPE specimens using eight different extraction kits and to evaluate the suitability of the DNA for methylation analysis using four different kits. Tumor-rich areas in four FFPE archival prostate specimens were cored (0.6 mm) and the resultant 120 cores were pooled, deparaffinized in two changes of xylene, washed, and suspended in ethanol. Specimens were then homogenized and aliquoted for extraction using each of the kits. Nucleic acids were extracted following the manufacturer’s instructions with the RecoverAll Total Nucleic Acid Isolation Kit for FFPE, AllPrep DNA/RNA FFPE Kit, GeneJET Genomic DNA Purification Kit, DNeasy Blood & Tissue Kit, QIAamp DNA FFPE Tissue, High Pure FPET DNA Isolation Kit, Norgen FFPE DNA Purification Kit, and NucleoSpin DNA FFPE XS kit. Nucleic acid yields were determined using the dsDNA HS (High Sensitivity) assay and purity was determined by spectrophotometric absorbance ratios. The presence of inhibitory substances was examined by real-time PCR amplification of the mouse HSD11β1 gene in cell-line DNA spiked with the extracted DNA. The maximum amplifiable fragment length of DNA was determined by amplifying 102, 165, 225, and 300 bp fragments of β-2-microglobulin. The suitability of DNA extracted using the AllPrep,QIAamp, RecoverAll, DNeasy, and GenJet kits for methylation-specific PCR was assessed for the CpG islands of Glutathione S-Transferase Pi 1(GSTP1), ATP -binding Cassette subfamily B member 1 (ABCB1) and Ras-association domain family member 1 (RASSF1).

Summary of Findings:

The highest DNA yields were obtained after extraction with the DNeasy kit (1236.03 ng/mm³) followed by the QIAamp (980 ng/mm³), RecoverAll (767.65 ng/mm³), AllPrep (757.2 ng/mm³), and GeneJet (750.59 ng/mm³) kits. Lower yields were obtained using the HighPure DNA (536.53 ng/mm³), Norgen DNA (468.53 ng/mm³), and NucleoSpin DNA (429.71 ng/mm³) kits. The A260/A280 ratio for the specimen extracted using the AllPrep kit was 1.77 but were at or above 1.8 for all other methods. Specimens extracted with the RecoverAll, AllPrep, QIAamp and DNeasy kits had low A260/A230 ratios (<1.8), indicating the presence of organic contaminants. However, only the contaminants present after extraction with AllPrep had an inhibitory effect on the amplification of a mouse gene and this effect was eliminated when DNA was diluted 10-fold. Each of the five kits prioritized for further testing (AllPrep, QIAamp, RecoverAll, DNeasy, and GeneJet) allowed for amplification of all four PCR products (102-300 bp), but the bands were less intense when extracted with the DNeasy kit. The Cq values for the three genes assessed by methylation-specific real-time PCR were significantly higher when DNA was extracted with RecoverAll or QIAamp (P<0.05 for all) than when extracted with AllPrep but were comparable when extracted with DNeasy or GeneJet.

Biospecimens

Tissue - Prostate

Preservative Types

Formalin

Diagnoses:

Neoplastic - Carcinoma

Platform:

Analyte	Technology Platform
DNA	Fluorometry
DNA	Real-time qPCR
DNA	Spectrophotometry
DNA	Bisulfite conversion assay

Pre-analytical Factors:

Classification	Pre-analytical Factor	Value(s)
Analyte Extraction and Purification	Analyte isolation method	RecoverAll Total Nucleic Acid Isolation Kit for FFPE AllPrep DNA/RNA FFPE Kit GeneJET Genomic DNA Purification Kit DNeasy Blood & Tissue Kit Norgen FFPE DNA Purification Kit QIAamp DNA FFPE Tissue High Pure FPET DNA Isolation Kit NucleoSpin DNA FFPE XS kit
Real-time qPCR Specific	Targeted nucleic acid	B2M
Real-time qPCR Specific	Length of gene fragment	102 bp 165 bp 225 bp 300 bp

Study Purpose

This study investigated the effect of FFPE block storage duration on the reproducibility of yield and quality of nucleic acids extracted from FFPE prostate specimens using the AllPrep kit at three different laboratories. Nine cores were obtained from each of 12 FFPE prostate cancer specimens archived 7 to 15 years. Cores were pooled by specimen, deparaffinized in xylene, and homogenized together. Equal volumes of the homogenate were distributed to the three independent laboratories. RNA and DNA were extracted at each laboratory using the AllPrep kit following the manufacturer’s supplied protocol. Nucleic acid yields were determined using the dsDNA HS (High Sensitivity) and RNA BR (Broad-Range) assay kits. Expression analysis was investigated by hybridization to NanoString 48-plex Customer Assay Evaluation. Methylation-specific PCR was assessed for the CpG islands of Glutathione S-Transferase Pi 1 (GSTP1), ATP-binding cassette subfamily B member 1 (ABCB1) and Ras-association domain family member 1 (RASSF1).

Summary of Findings:

There was no effect of duration of FFPE block storage on the DNA yield, RNA yield, methylation-specific PCR Cq values or NanoString counts. DNA and RNA yields and purity were comparable among the three laboratories performing the extractions. Further, methylation-specific PCR Cq and NanoString counts were very strongly correlated between the laboratories (r=0.9679-0.9834 and r=0.9979-0.998, respectively).

Biospecimens

Tissue - Prostate

Preservative Types

Formalin

Diagnoses:

Neoplastic - Carcinoma

Platform:

Analyte	Technology Platform
DNA	Fluorometry
DNA	Spectrophotometry
DNA	Bisulfite conversion assay
RNA	DNA microarray
RNA	Spectrophotometry
RNA	Real-time qRT-PCR
RNA	Fluorometry

Pre-analytical Factors:

Classification	Pre-analytical Factor	Value(s)
Analyte Extraction and Purification	Analyte isolation method	Three independent laboratories
Storage	Storage duration	6 years 7 years 8 years 10 years 12 years 13 years 15 years

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