Evaluation and Validation of Total RNA Extraction Methods for MicroRNA Expression Analyses in Formalin-Fixed, Paraffin-Embedded Tissues
2008; Elsevier BV; Volume: 10; Issue: 3 Linguagem: Inglês
10.2353/jmoldx.2008.070153
ISSN1943-7811
AutoresMartina Doleshal, Amber A. Magotra, Bhavna Choudhury, Brian D. Cannon, Emmanuel Labourier, Anna E. Szafranska,
Tópico(s)Cancer-related molecular mechanisms research
ResumoHistopathology archives of well-annotated formalin-fixed, paraffin-embedded (FFPE) tissue specimens are valuable resources for retrospective studies of human diseases. Since recovery of quality intact mRNA compatible with molecular techniques is often difficult due to degradation, we evaluated microRNA (miRNA), a novel class of small RNA molecules with growing therapeutic and diagnostic potential, as an alternative analyte for gene expression studies of FFPE samples. Analyzing total RNA yield, miRNA recovery, and robustness of real-time polymerase chain reaction for miRNA, mRNA, and rRNA species, we compared the performance of commercially available RNA isolation kits and identified a preferred methodology. We further implemented modifications to increase tissue throughput and incorporate a quantitative Armored RNA process control to monitor RNA recovery efficiency. The optimized process was tested for reproducibility as well as interoperator and interday variability, and was validated with a set of 30 clinical samples. In addition, we demonstrated that, independent of FFPE block age and RNA quality, miRNAs generate quantitative reverse transcription-polymerase chain reaction signals that are more robust and better correlate with expression levels from frozen reference samples compared with longer mRNAs. Our broad study, including a total of 272 independent RNA isolations from 17 tissue types and 65 FFPE blocks up to 12 years old, indicates that miRNAs are not only suitable but are also likely superior analytes for the molecular characterization of compromised archived clinical specimens. Histopathology archives of well-annotated formalin-fixed, paraffin-embedded (FFPE) tissue specimens are valuable resources for retrospective studies of human diseases. Since recovery of quality intact mRNA compatible with molecular techniques is often difficult due to degradation, we evaluated microRNA (miRNA), a novel class of small RNA molecules with growing therapeutic and diagnostic potential, as an alternative analyte for gene expression studies of FFPE samples. Analyzing total RNA yield, miRNA recovery, and robustness of real-time polymerase chain reaction for miRNA, mRNA, and rRNA species, we compared the performance of commercially available RNA isolation kits and identified a preferred methodology. We further implemented modifications to increase tissue throughput and incorporate a quantitative Armored RNA process control to monitor RNA recovery efficiency. The optimized process was tested for reproducibility as well as interoperator and interday variability, and was validated with a set of 30 clinical samples. In addition, we demonstrated that, independent of FFPE block age and RNA quality, miRNAs generate quantitative reverse transcription-polymerase chain reaction signals that are more robust and better correlate with expression levels from frozen reference samples compared with longer mRNAs. Our broad study, including a total of 272 independent RNA isolations from 17 tissue types and 65 FFPE blocks up to 12 years old, indicates that miRNAs are not only suitable but are also likely superior analytes for the molecular characterization of compromised archived clinical specimens. Since the initial development of standardized methodology for fixation and processing of human tissue samples at the turn of the last century, anatomical pathology practices around the world have been preserving biopsies and other pathological specimens as formalin-fixed, paraffin-embedded (FFPE) blocks. This process has resulted in a widely available and rich archive of well characterized tissue specimens annotated with patient data, which offer a valuable source of information for comparative genomics as well as for biomarker discovery studies.1Lewis F Maughan NJ Smith V Hillan K Quirke P Unlocking the archive–gene expression in paraffin-embedded tissue.J Pathol. 2001; 195: 66-71Crossref PubMed Scopus (260) Google Scholar With the advent of high-content, high-throughput molecular genetic techniques such as quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and microarrays, there has been growing interest in mining these archival collections as a source of biological data. While the value of retrospective pathological analysis of archived tissues has been extensively validated, until very recently these samples have not been considered reliable sources of mRNA for gene expression studies due to the difficulty in obtaining intact mRNA from these samples. Therefore optimization of the recovery of quality RNA from FFPE tissues is of particular interest to many research laboratories.2Coombs NJ Gough AC Primrose JN Optimisation of DNA and RNA extraction from archival formalin-fixed tissue.Nucleic Acids Res. 1999; 27: e12Crossref PubMed Google Scholar,3Chung JY Braunschweig T Hewitt SM Optimization of recovery of RNA from formalin-fixed, paraffin-embedded tissue.Diagn Mol Pathol. 2006; 15: 229-236Crossref PubMed Scopus (75) Google ScholarThe procedure for preserving and archiving tissues involves fixation of the tissue in formalin (10% formaldehyde) or ethanol-based preservatives followed by processing by dehydration with graded ethanol solutions to enhance the infusion of the material with paraffin. While formalin fixation helps preservation of cellular proteins and conserves the tissue architecture, it significantly reduces the recovery and quality of RNA. Extensive cross-linking of RNA with proteins during fixation renders RNA more resistant to extraction. Enzyme degradation, which occurs before and during the fixation process, as well as chemical degradation, results in decreased yield and integrity of RNA. Finally, formalin is responsible for forming mono-methylol adducts with bases of nucleic acids, in particular with adenine.4Masuda N Ohnishi T Kawamoto S Monden M Okubo K Analysis of chemical modification of RNA from formalin-fixed samples and optimization of molecular biology applications for such samples.Nucleic Acids Res. 1999; 27: 4436-4443Crossref PubMed Scopus (521) Google Scholar,5Korga A Wilkolaska K Korobowicz E Difficulties in using archival paraffin-embedded tissues for RNA expression analysis.Postepy Hig Med Dosw (online). 2007; 61: 151-155PubMed Google Scholar This covalent modification reduces the efficiency of reverse transcription in qRT-PCR and negatively affects the performance of RNA samples in other downstream applications. Recently, many research groups have attempted to overcome these limitations. The effects of new fixatives on histological properties of tissues as well as on the quality and yield of RNA have been tested.6Benchekroun M DeGraw J Gao J Sun L von Boguslawsky K Leminen A Andersson LC Heiskala M Impact of fixative on recovery of mRNA from paraffin-embedded tissue.Diagn Mol Pathol. 2004; 13: 116-125Crossref PubMed Scopus (52) Google Scholar7Stanta G Mucelli SP Petrera F Bonin S Bussolati G A novel fixative improves opportunities of nucleic acids and proteomic analysis in human archive's tissues.Diagn Mol Pathol. 2006; 15: 115-123Crossref PubMed Scopus (85) Google Scholar8Gillespie JW Best CJ Bichsel VE Cole KA Greenhut SF Hewitt SM Ahram M Gathright YB Merino MJ Strausberg RL Epstein JI Hamilton SR Gannot G Baibakova GV Calvert VS Flaig MJ Chuaqui RF Herring JC Pfeifer J Petricoin EF Linehan WM Duray PH Bova GS Emmert-Buck MR Evaluation of non-formalin tissue fixation for molecular profiling studies.Am J Pathol. 2002; 160: 449-457Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar9Paska C Bogi K Szilak L Tokes A Szabo E Sziller I Rigo Jr, J Sobel G Szabo I Kaposi-Novak P Kiss A Schaff Z Effect of formalin, acetone, and RNAlater fixatives on tissue preservation and different size amplicons by real-time PCR from paraffin-embedded tissue.Diagn Mol Pathol. 2004; 13: 234-240Crossref PubMed Scopus (61) Google Scholar Methods that do not require RNA extraction, such as in situ hybridization, have also been evaluated as a novel approach to gene expression analysis in FFPE tissues,10Henke RT Maitra A Paik S Wellstein A Gene expression analysis in sections and tissue microarrays of archival tissues by mRNA in situ hybridization.Histol Histopathol. 2005; 20: 225-237PubMed Google Scholar11Henke RT Eun Kim S Maitra A Paik S Wellstein A Expression analysis of mRNA in formalin-fixed, paraffin-embedded archival tissues by mRNA in situ hybridization.Methods. 2006; 38: 253-262Crossref PubMed Scopus (20) Google Scholar12To MD Done SJ Redston M Andrulis IL Analysis of mRNA from microdissected frozen tissue sections without RNA isolation.Am J Pathol. 1998; 153: 47-51Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar but they are not amenable to high-content, high-throughput analyses. To this day, no alternative fixative has replaced formalin fixation as a preservation method in routine clinical use.In the past seven years there has been an explosion of interest in miRNAs (microRNAs) with many hundreds of publications describing the fundamental role these regulatory biomolecules play in processes as diverse as early development, cell proliferation, differentiation, apoptosis, and oncogenesis.13Lee RC Ambros V An extensive class of small RNAs in Caenorhabditis elegans.Science. 2001; 294: 862-864Crossref PubMed Scopus (2290) Google Scholar14Lagos-Quintana M Rauhut R Lendeckel W Tuschl T Identification of novel genes coding for small expressed RNAs.Science. 2001; 294: 853-858Crossref PubMed Scopus (3892) Google Scholar15Calin GA Ferracin M Cimmino A Di Leva G Shimizu M Wojcik SE Iorio MV Visone R Sever NI Fabbri M Iuliano R Palumbo T Pichiorri F Roldo C Garzon R Sevignani C Rassenti L Alder H Volinia S Liu CG Kipps TJ Negrini M Croce CM A MicroRNA signature associated with prognosis and progression in chronic lymphocytic leukemia.N Engl J Med. 2005; 353: 1793-1801Crossref PubMed Scopus (2072) Google Scholar16Esquela-Kerscher A Slack FJ Oncomirs—microRNAs with a role in cancer.Nat Rev Cancer. 2006; 6: 259-269Crossref PubMed Scopus (6132) Google Scholar17Volinia S Calin GA Liu CG Ambs S Cimmino A Petrocca F Visone R Iorio M Roldo C Ferracin M Prueitt RL Yanaihara N Lanza G Scarpa A Vecchione A Negrini M Harris CC Croce CM A microRNA expression signature of human solid tumors defines cancer gene targets.Proc Natl Acad Sci USA. 2006; 103: 2257-2261Crossref PubMed Scopus (4893) Google Scholar Due to their small size (19 to 23 nucleotides), miRNAs may be less prone to degradation and modification, and therefore their analysis in FFPE specimens likely provides a more accurate replication of what would be observed in fresh tissue than that of mRNA species. Although several RNA isolation protocols or commercial kits have been optimized for miRNA recovery, no detailed analysis of their relative performance has been performed. In this study we compared different commercially available isolation procedures by evaluating the yield and quality of isolated total RNA as well as the detection efficiency of miRNA, mRNA, and rRNA species. We identified and validated a robust protocol using multiple tissue types and FFPE blocks with ages ranging from 1 to 12 years. We also introduced new procedural improvements to facilitate the increase in tissue throughput as well as to enable better control over the extraction process. Our results suggest that with the appropriate RNA isolation protocol and controls, miRNAs are excellent candidates for biomarker discovery studies using archived clinical specimens.Materials and MethodsTissue SamplesSamples derived from human patients were acquired from commercial suppliers by Asuragen's Tissue Procurement Group in compliance with the regulations as outlined in Title 45 of the Code of Federal Regulations, Part 46 (45 CFR 46) and other regulatory guidance. All samples used in this study were collected as part of standard clinical care and were considered to be remnant materials unnecessary for patient treatment. Patient identifiers were thoroughly removed from all samples before distribution to Asuragen. Samples used in the comparison between flash-frozen and FFPE tissues included cervical, breast, and gall bladder from two or three donors for each tissue. Half of each tissue was flash-frozen and half was formalin-fixed and embedded into paraffin according to a standard fixation procedure (<60 minutes elapsed time between surgery and immersion in 10% neutral buffered formalin for 24 hours). Older FFPE tissue blocks, aged from 3 to 12 years, also interrogated in this study included breast cancer, lung cancer, normal kidney, normal cervix, testicular cancer, stomach cancer, uterine cancer, lung normal adjacent tissue (NAT), prostate NAT, spleen, and liver NAT.RNA IsolationFor the initial evaluation of isolation kits, total RNA from two FFPE tissues blocks, breast tumor and lung tumor, was isolated using five commercially available kits, RNeasy FFPE Kit (Qiagen, Valencia, CA), Absolutely RNA FFPE Kit (Stratagene, La Jolla, CA), High Pure FFPE RNA Micro Kit (Roche, Indianapolis, IN), PureLink FFPE RNA Isolation Kit (Invitrogen, Carlsbad, CA) and RecoverAll Total Nucleic Acid Isolation Kit (Ambion, Austin, TX), according to the respective manufacturers' instructions. In the case of the RNeasy FFPE Kit, a supplementary protocol for "co-purification" of total RNA and miRNA from FFPE tissue sections using the RNeasy FFPE Kit was used. Total RNA from a subset of FFPE blocks, including breast cancer, lung cancer, lung NAT, prostate NAT, spleen, and liver NAT was isolated using RecoverAll and RNeasy kits. Total RNA from remaining tissue blocks (normal kidney, normal cervix, testicular cancer, stomach cancer, and uterine cancer) was isolated using RecoverAll protocol only. To accommodate higher FFPE tissue input, the later protocol was modified based on results of the following experiment. Two prostate cancer FFPE blocks (1 year old), differing in size of cross-sectional tissue area ( 150 mm2), were cut into 20-μm slices to accommodate four, eight, 12, and 16-slice isolations in duplicate. All slices were placed in a 15-ml conical tube and deparaffinized together (six 10-ml washes with 100% xylene, followed by three 10-ml washes with 100% ethanol, and dried in a Speedvac at 30°C for ∼10 minutes). The dry tissue was resuspended in 4.8 ml of proteinase K buffer and homogenized using a PRO 250 tissue homogenizer (PROScientific, Oxford, CT). The tissue homogenate was aliquoted in volumes corresponding to four-, eight-, 12-, and 16-slice proteinase K tissue digestion (400, 800, 1200, and 1600 μl). On addition of customized proteinase K volume to each tube, the proteinase K digestion and remaining steps were performed as per the manufacturer's protocol.Total RNA from frozen tissues was extracted using mirVana miRNA Isolation Kit (Ambion) following the manufacturer's protocol. Concentration and purity of the total RNA samples were measured using the NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE). RNA integrity was assessed with an Agilent 2100 Bioanalyzer and the RNA 6000 LabChip kit (Agilent Technologies, Palo Alto, CA).Real-Time Quantitative RT-PCRThe recovery of miRNA and mRNA species was estimated via quantification of relative expression levels of miR-24, miR-103, miR-191, 18S, and hGUSB. qRT-PCR experiments were performed using 10 ng total RNA input and TaqMan primer/probe sets (TaqMan Gene Expression Assays, Applied Biosystems, Foster City, CA). For miRNA amplification, a two-step qRT-PCR was performed as follows: RT in duplicate (16°C, 30 minutes; 42°C, 30 minutes; 85°C, 5 minutes, then hold at 4°C); PCR in duplicate from each RT (95°C, 1 minute; 95°C, 15 seconds followed by 60°C, 30 seconds and cycled 40 times). For mRNA amplification, a one-step qRT-PCR was performed in duplicate under the following conditions: 42°C, 15 minutes; 95°C, 2 minutes; then 95°C, 15 seconds followed by 60°C, 45 seconds and cycled 40 times. PCR amplifications were performed on a 7900HT Fast real-time PCR system and data analyzed with the 7900HT Fast system SDS software v2.3 (Applied Biosystems).Implementation of an Internal Isolation Process ControlA total of 1010 copies of Armored RNA Quant (ARQ) (Asuragen, Austin, TX) containing a non-human RNA sequence was spiked in during RNA isolation from one or four 20-μm slices of FFPE tissue, either at the proteinase K digestion step or at the RNA elution step. One-step qRT-PCR was performed using ARQ-specific primers and probe to quantify the percentage of ARQ recovery. Each qRT-PCR reaction was run in duplicate, including a positive control ARQ heat-lysed for 3 minutes at 95°C in nuclease-free water and a no template control. For extraction from 16 slices (20-μm thickness) of FFPE lung tumor and NAT blocks, ARQ was spiked at the proteinase K digestion step and recovery was assessed as described above.ResultsMethod Comparison for RNA Extraction from FFPE TissuesAs a first step toward identifying the most suitable procedure for RNA isolation from FFPE tissues, we isolated total RNA in duplicate from two tissue types, lung cancer and breast cancer, using five different commercially available isolation procedures: Absolutely RNA FFPE Kit, High Pure FFPE RNA Micro Kit, PureLink FFPE RNA Isolation Kit, RecoverAll Total Nucleic Acid Isolation Kit, and RNeasy FFPE Kit. This comparison was based on criteria such as yield of total RNA and efficient recovery of selected miRNA and mRNA species (data not shown). Three kits showed two- to threefold lower total RNA yield and five- to 20-fold lower miRNA qRT-PCR signals at equal RNA mass input, and were not further evaluated. The two RNA isolation kits that demonstrated superior performance, RecoverAll and RNeasy, were subjected to a more comprehensive characterization study.For a thorough comparison of the two isolation kits, total RNA was isolated from 13 FFPE blocks that ranged in age from 1 to 12 years and comprised seven distinct tissues: normal breast, normal cervix, normal gall bladder, "normal" lung tissue from lung cancer patients (normal adjacent tissue or NAT), prostate NAT, liver NAT, and normal spleen. The comparison of average RNA recovery from triplicate isolations revealed higher yields in samples isolated with RecoverAll by an average of 1.7-fold (Table 1). Eleven of the 13 FFPE blocks analyzed (55% to 98% of the samples with 95% confidence) generated lower RNA yields with the RNeasy protocol. In addition, within each FFPE block, the final RNA amount recovered was more consistent when RecoverAll was used, with %CV for triplicate isolations ranging from 5% to 28%, versus 10% to 43% for isolations performed with RNeasy. Agilent Bioanalyzer electropherograms showed roughly similar RNA integrity for both kits (data not shown). However, RNA purity was greater with RecoverAll, as demonstrated by an average A260/280 ratio of 1.98 for RecoverAll isolations versus 1.68 for RNeasy isolations (P = 0.0003) (Table 1).Table 1Summary of Triplicate RNA Isolations from 13 FFPE Tissue Blocks Using the RecoverAll or RNeasy ProcedureRecoverAllRNeasyAvg RNA yield (μg)A260/A280Avg RNA yield (μg)A260/A280Tissue1 × 20 μmCV (%)Avg ratioCV (%)2 × 10 μmCV (%)Avg ratioCV (%)Breast-10.1511.02.0116.40.0943.11.545.7Breast-20.235.12.016.20.1639.01.6710.1Breast-30.1825.52.088.70.1142.61.533.9Cervix-10.6918.12.066.31.619.82.066.5Cervix-20.6314.02.032.20.3914.41.506.3Cervix-30.6528.21.967.70.4710.71.463.5Gall bladder-10.3414.11.947.70.1126.61.465.0Gall bladder-20.317.41.8822.00.4411.31.413.9Lung NAT-12.6327.91.960.51.9516.01.881.3Lung NAT-21.168.91.983.10.8433.11.726.1Prostate NAT2.4414.01.890.61.4717.01.721.2Spleen4.4212.01.990.93.4226.91.930.9Liver NAT-15.7617.21.932.73.3736.51.931.4 Open table in a new tab Quantitative Gene Expression AnalysesTo evaluate the quality and efficiency of miRNA and mRNA extraction from FFPE tissues we next interrogated expression levels of miR-24, miR-191, miR-103, 18S rRNA, and hGUSB mRNA by qRT-PCR in a representative set of FFPE RNA samples (two lung NAT, one prostate NAT, one liver NAT, and one spleen) using TaqMan primers and probes and equal input of total RNA for each sample (10 ng). miR-24 and miR-103 were detected at higher levels in the FFPE samples isolated with RecoverAll, 2.2-fold (P = 0.06) and 2.1-fold (P = 0.07) on average, respectively, while detection of miR-191 was comparable for both kits (Figure 1). The performance difference between kits was more striking for larger RNA species, although P values were not significant due to high amplification variability between tissue types. The 18S and the hGUSB were detected 11.7-fold (P = 0.2) and 2.4-fold (P = 0.3) more abundantly in RNA samples isolated with RecoverAll. No amplification signals were obtained when reverse transcriptase or RNA was omitted (data not shown).Comparison with Matched Frozen TissuesTo further evaluate each method we also compared RNA samples isolated from multiple breast, cervix, and gall bladder blocks, with RNA samples isolated from the corresponding paired flash-frozen tissues using a method optimized for small RNA recovery (see Materials and Methods). As expected, the quality of RNA isolated from frozen tissues was high, with distinct 18S and 28S rRNA bands, while RNA isolated from fixed tissues displayed a low molecular weight distribution, with an average size lower than 100 nucleotides, regardless of the isolation kit used (see Supplemental Figure S1 at http://jmd.amjpathol.org).Analysis of miR-24, miR-103, and miR-191 expression levels by qRT-PCR showed a significant difference between frozen and FFPE samples with the average cycle threshold (Ct) notably higher in RNA samples isolated from FFPE specimens (Figure 2A; P = 2 × 10−4 for RNeasy and P = 5.5 × 10−7 for RecoverAll). miRNA detection in cervical FFPE samples more closely matched the reference levels from frozen samples when the RNeasy kit was used (P = 0.003; Figure 2C). In contrast, the use of RecoverAll resulted in better miRNA detection in total RNA samples from breast and gall bladder tissues (P = 8.0 × 10−6). The ΔCt between frozen and FFPE samples was on average 0.65 Ct lower (1.6-fold higher miRNA representation) in RecoverAll samples relative to RNeasy samples. For longer RNA species, detection of 18S and hGUSB targets by qRT-PCR in RNA samples from FFPE blocks relative to paired reference frozen tissues were improved on average by 2.45 Ct or 5.5-fold (P = 0.05) with RecoverAll relative to RNeasy (Figure 2, B and C).Figure 2Comparison between FFPE and matching frozen samples. A: Average expression levels for miR-24, -103, and -191 in RNA samples isolated from FFPE and matched frozen breast (three specimens), cervix (three specimens), and gall bladder (two specimens) tissues. Each qRT-PCR reaction was performed in duplicate. B: Same as A for 18S rRNA and hGUSB mRNA. C: Average differential expression (ΔCt) between frozen and matching FFPE samples for miRNAs or mRNA and rRNA species. D: Average standard deviation of qRT-PCR signal (Ct) within the 13 FFPE tissues blocks described in Table 1 for miRNA or mRNA and rRNA species.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Finally, we examined the overall variability in RNA amplification efficiency within the 13 different FFPE blocks tested so far (Figure 2D). We observed that RecoverAll isolation procedure provided a more consistent miRNA, mRNA, and rRNA qRT-PCR signal across all FFPE blocks and tissue types, with a standard deviation on average 1.5-fold lower than with RNeasy. Based on more robust and overall superior performance of the kit we selected RecoverAll as our preferred RNA isolation method and used this kit for the remainder of the study.Qualification of the RNA Extraction MethodWith the recommended RecoverAll protocol (isolation from a maximum of four 20-μm FFPE tissue slices), some tissue types with large cross-sectional area, such as spleen and liver NAT, yielded higher total RNA amounts than other specimens with smaller cross-sectional area, such as normal breast and skin (Table 2). To enable isolation of RNA from FFPE tissue blocks with various cross-sectional areas in quantities sufficient for multiple expression analyses and archiving of the purified RNA, we first determined the maximum number of FFPE slices that could be processed in a single tube. Isolations were performed in duplicate using 4, 8, 12, or 16 slices of FFPE tissue (20-μm thickness) from two prostate cancer FFPE blocks that differed in their tissue cross-sectional area (see Materials and Methods). For the tissue block with a smaller cross-sectional area ( 150 mm2), not all of the tissue was digested in tubes containing more than four 20-μm slices resulting in yields that were lower than expected (data not shown). However, by increasing the amount of proteinase K added to the digestion, we improved the yields by 40% and rescued the linearity of recovery up to 12 slices. For blocks with both large and small cross-sectional areas we were able to recover over 30 μg of purified total RNA in a single isolation (Figure 3).Table 2Summary of Total RNA Yields Recovered from 17 Different Tissue Types Representing a Total of 65 FFPE Blocks and 272 Independent RNA Isolations Using the RecoverAll ProcedureFFPE tissueNo. of isolationsNo. of blocksBlock age (yr)Avg RNA yield (μg) per 20-μm sliceCV (%)B-cell lymphoma6212.7232.4Breast9310.1921.4Cervix2041–30.4946.9Colon5212.5532.3Colon cancer4131.7522.2Gall bladder6210.336.1Kidney121120.976.1Liver NAT81123.872.5Lung NAT70171–111.3153.4Lung cancer53151–112.7170.1Myometrium2891–111.8244.5Prostate NAT3181.6713.9Skin631–30.2161.9Spleen6183.1159.3Stomach cancer12191.2811.5Testicular cancer12141.674.0Uterine cancer121101.115.2 Open table in a new tab Figure 3Scale-up of the RecoverAll procedure. RNA isolation was performed with four, eight, 12, or 16 FFPE tissue slices (20-μm) from two prostate cancer FFPE blocks that differed in tissue cross-sectional area. For the block with a tissue area >150 mm2, twice the recommended amount of proteinase K was used.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To determine the reproducibility of this method, we performed repeated isolations using four 20-μm slices from two different FFPE blocks: liver NAT (five independent isolations) and spleen (three independent isolations). The purified RNA samples were compared in terms of yield, purity, miRNA, rRNA, and mRNA amplification by qRT-PCR. The variation in RNA recovery from individual blocks was less than 21% for liver NAT and less than 5% for spleen (Table 3). The A260/280 ratio was highly reproducible, ranging from 1.79 to 1.84 for liver NAT and from 1.91 to 1.94 for spleen. The quantification of miR-24, miR-103, miR-191, 18S, and hGUSB panel by qRT-PCR was also highly reproducible with %CV across all samples less than 2.8% (Table 3).Table 3Summary of Method Variability DataRNA isolationTarget amplificationAvg yieldA260/280miR-24miR-103miR-19118ShGUSBTissueμgCV (%)Avg ratioCV (%)Avg CtCV (%)Avg CtCV (%)Avg CtCV (%)Avg CtCV (%)Avg CtCV (%)Liver NAT7.4220.71.801.125.522.427.581.426.112.715.932.429.051.6Spleen7.234.51.921.123.862.825.612.124.052.412.912.325.921.2Five and three independent RNA isolations using four 20-μm slices from a liver NAT and spleen blocks, respectively, were performed by the same operator on the same day using the RecoverAll procedure. qRT-PCR reactions were performed at least in duplicate for each RNA target. Open table in a new tab We next investigated interoperator variability with respect to RNA yield as well as extraction efficiency of the miRNA, rRNA, and mRNA fractions. Total RNA from five different FFPE tissue types ranging from 3 to 12 years old was extracted in duplicate by four different operators on the same day (Table 4). The %CV between operators ranged from 7.7% to 25% for yield, and 8.6% to 21.2% for A260/280 ratio. RNA quantification by qRT-PCR was very reproducible with %CV ranging from 0.2% to 6.4% and no significant difference among miRNA, mRNA, and rRNA species. A clear trend was observed between tissue types with cervix generating the highest %CV, while uterine cancer replicates were very consistent in terms of RNA yield, quality, and amplification efficiency. Two operators also performed duplicate RNA isolation from the five different tissues on a second day. On average, day-to-day variability was 20% for RNA yield and %CV for qRT-PCR amplification was lower than 2% for all interrogated targets (see Supplemental Table S1 at http://jmd.amjpathol.org).Table 4Summary of Interoperator Variability DataRNA isolationTarget amplificationAv yieldA260/A280miR-24miR-103miR-19118ShGUSBFFPE tissueμgCV (%)Avg ratioCV (%)Avg CtCV (%)Avg CtCV (%)Avg CtCV (%)Avg CtCV (%)Avg CtCV (%)Cervix0.622.72.2021.222.682.628.182.327.002.217.742.830.803.7Kidney1.923.52.166.623.256.426.801.726.230.917.091.727.111.4Uterine cancer2.37.72.138.623.670.426.210.724.810.717.990.526.400.2Testicular cancer3.38.32.249.222.652.227.650.626.191.214.530.426.661.3Stomach cancer2.325.02.1112.922.791.526.291.826.220.617.452.026.160.6Duplicate RNA isolations using two 20-μm slices from five different FFPE tissue blocks were performed by four different operator
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