Comparison of DNA and RNA from fresh-frozen vs. FFPE tissue samples

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1 PPLICTION NOTE PE DN/RN Comparison of DN and RN from fresh-frozen vs. PE tissue samples Key findings The pplied iosystems PE DN/RN provides fast, reliable sequential nucleic acid isolation from formalin-fixed, paraffin-embedded (PE) samples. The quality, purity, and yield obtained from PE samples can be very comparable to their matched fresh-frozen () counterparts, especially when the PE samples were controlled under standardized conditions, as outlined later. The process of fixing and embedding the PE samples does not render their nucleic acids useless for genomic analyses, and they can suitably serve as templates. Regardless of the sample type, i.e., PE or, the same conclusions could be reached in terms of variant calling and gene expression. Introduction Extraction of nucleic acids from and PE tissues is a critical step in routine workflows for biomedical researchers. Extraction of both DN and RN from a single section of a tissue sample in a single workflow can now be achieved with the PE DN/RN using magnetic separation techniques. To demonstrate that the extracted nucleic acids are pure and highly functional, they were compared to those obtained using other kits that are designed and optimized to only extract DN or RN from samples. ll samples were processed on the Thermo Scientific HM 3S utomatic Microtome, the Thermo Scientific CryoStar NX7 cryostat, and the Thermo Scientific KingFisher Flex Purification System, which enables high-throughput, robust, and repeatable nucleic acid extraction from and PE tissues. In addition to quantitating the samples, we also analyzed RN integrity, performed real-time PCR to assess functionality of the nucleic acids, and sequenced samples to evaluate hotspot regions in commonly mutated human cancer genes and their corresponding RN transcripts. Materials and methods Tissue selection and PE preparation Nucleic acids were extracted from cancerous human tissues purchased from sterand ioscience (Detroit, MI). ll tissues used conformed to the following criteria: de-identified patient information, >2 g of sample with >9% tumor volume, and RN integrity number (RIN) >9.. Two different tissue types with significant clinical relevance were selected for this study: lung cancer samples from non-small cell lung carcinoma (LCa) of adenocarcinoma subtype from three different patients, and breast cancer (Ca) samples from infiltrating ductal adenocarcinoma tissues from three different patients. ll frozen tissues were kept at 8 C except during grossing and sectioning, and immediately returned to 8 C when possible. One section was saved from each tissue sample to serve as a control sample, and the rest were fixed and embedded. Tissues were fixed in 1% Thermo Scientific Richard- llan Scientific Neutral uffered Formalin (NF) for 24 hours, sectioned into 3 mm 3 pieces, then processed for 8 hours on a Thermo Scientific Excelsior ES Tissue Processor, and embedded in Thermo Scientific Richard- llan Scientific Histoplast Paraffin at a Thermo Scientific HistoStar Embedding Workstation.

2 Fixation, processing, and embedding took place under RNase-minimized conditions using tools and surfaces sterilized with 7% ethanol in nuclease-free water, followed by rinsing with Invitrogen RNase WY Decontamination Reagent. ll of the blocks were prepared in 216. Instrumentation and kits for tissue sectioning and nucleic acid extraction The tissues were sectioned at 2 C on a CryoStar NX7 cryostat, cutting 7 µm curls. single curl was placed into µl of respective lysis buffer with cold forceps. Tubes were kept inverted on ice until all sectioning was complete. fter sectioning, all tubes were centrifuged at 12, rpm at 4 C for 1 minute to collect the tissue and buffer at the bottom of the Thermo Scientific Sorvall Legend Micro 21 microcentrifuge tube. Then the remaining lysis buffer was added. The PE tissues were sectioned on an HM 3S utomatic Microtome set at a 7 µm cutting thickness using Thermo Scientific MX3 Premier Disposable Low-Profile Microtome lades. single curl was placed directly into an empty sterile 1. or 2. ml microcentrifuge tube. The PE samples were then deparaffinized using a standard xylene protocol as outlined in the PE DN/RN. The KingFisher Flex Purification System was used with the following compatible kits for lysate preparation and nucleic acid extraction: pplied iosystems mirvana Total RN Isolation Kit (for RN from tissue), DN Multi-Sample Kit (for DN from tissue), and PE DN/RN (for both DN and RN from PE tissue). Methods for these kits were carried out according to the protocols listed in their manuals, using Thermo Scientific KingFisher Flex sterile microtiter 96 deep-well plates. Nucleic acid quantitation and RN analysis Extracted RN and DN were quantified using the Invitrogen Qubit 3. Fluorometer using the Qubit dsdn and RN HS ssay Kits, and the Thermo Scientific NanoDrop 2c Spectrophotometer. Nucleic acid purity was measured on the NanoDrop spectrophotometer, focusing on 26 / 23 and 26 / 28 absorbance ratios. RN fragment sizes were measured using an gilent Technologies 21 ioanalyzer system. One sample from each group was run on the ioanalyzer system. Real-time PCR assays For the DN samples, DN inputs of equal mass were used in 1 µl PCR reactions with the pplied iosystems TaqMan Universal II Master Mix, no UNG, and run on the pplied iosystems 79HT Fast Real-Time PCR System with 384-well block under standard cycling conditions. iological duplicates from each of the three donors and for each tissue type were processed. For the RN samples, RN inputs of equal mass were used in 2 µl reverse-transcription reactions. The Invitrogen SuperScript VILO cdn Synthesis Kit was used for the generation of first-strand cdn under standard cycling conditions. Two microliter from the reverse transcription reaction was used in a 1 µl PCR reaction with the TaqMan Universal II Master Mix, no UNG, and run on the 79HT Fast Real-time PCR System with 384-well block under standard cycling conditions. iological duplicates from each of the three donors and for each tissue type were processed. Next-generation targeted sequencing Ten nanograms of each DN and RN was used in the Ion mpliseq Library Kit 2. with the Ion mpliseq Cancer Hotspot Panel v2 and the Ion mpliseq RN Cancer Panel, with the appropriate number of cycles. Libraries were then quantitated with the Ion Library TaqMan Quantitation Kit. ased on the quantitation, libraries were pooled at equimolar concentration and diluted to 6 pm for the Ion Chef instrument, and samples were then sequenced on an Ion 3 chip using the Ion S sequencing system. Data were analyzed with the Torrent Suite Coverage nalysis plugin, Torrent Variant Caller plugin, and the mpliseqrn plugin. Results and discussion The Qubit fluorometer and the NanoDrop spectrophotometer are instruments for nucleic acid quantitation, with their own advantages and disadvantages. Due to specific dye-binding chemistry, the Qubit assays are sensitive enough to distinguish between RN and DN species, whereas the NanoDrop instrument measures all absorbance at 26 nm. Since RN, ssdn, and dsdn all absorb at 26 nm, the NanoDrop measurements tend to overestimate nucleic acid concentrations.

3 The nucleic acid yields varied from donor to donor, as expected. On average, the Ca samples yielded over 12 ng of DN and 2 ng of RN. For PE Ca samples, average yields were about 1 ng of DN and 7 ng of RN. For LCa samples, 1 ng of DN and ng of RN were obtained. For PE LCa samples, average yields were about 1 ng of DN and 7 ng of RN (Figure 1). lthough the PE RN is fragmented, sufficient yield was obtained, but the template may not be as pristine. dditionally, through the fixation process RNases are inactivated in PE samples, whereas frozen tissue still contains RNases, which become active as they thaw, which may be contributing to lower yield in the RN samples. The PE Ultra kit was able to retrieve a quantity of DN from PE samples similar to or greater than the amount of DN obtained from sections using the DN Multi-Sample Kit. vg RN yield (ng) vg DN yield (ng) 2, 2, 1, 1, 4, 4, 3, 3, 2, 2, 1, 1, DN Multi-Sample DN yield average of 3 donors PE DN/RN cancer sample mirvana Total RN Isolation Kit DN Multi-Sample PE DN/RN cancer sample RN yield average of 3 donors PE DN/RN cancer sample mirvana Total RN Isolation Kit PE DN/RN cancer sample Qubit NanoDrop Qubit NanoDrop Figure 1. verage DN and RN yields from 3 donor samples. () DN and () RN from samples were extracted using the DN Multi-Sample and mirvana Total RN Isolation Kit, respectively; the PE DN/RN was used to extract both DN and RN from PE samples. For each sample type, one 7 µm section was used. The and PE samples were quantified using the Qubit and NanoDrop instruments, respectively. The ratio of absorbance at 26 nm and 28 nm ( 26 / 28 ) is used to assess the purity of DN and RN. In cases where there are low readings using the NanoDrop spectrophotometer (<1 ng/µl), ratio readings are skewed and less accurate. PE RN 26 / 28 values were at the optimal ratio of 2., but the samples were slightly outside the range. 26 / 28 values for DN were close to the optimal range for PE sections extracted using the PE Ultra kit, but the values for DN were slightly higher than optimal (Figure 2). secondary purity measurement is the 26 / 23 ratio. The 26 / 23 ratios for DN were low, which is attributable to the low nucleic acid concentrations, so accurate 26 / 23 ratios could not be obtained for DN, but for RN these ratios were higher. lthough purity ratios and spectral profiles can be indicators of sample quality, the best indicator of quality is the appropriate function of the nucleic acid samples in your desired downstream applications. With the achieved purity ratios, there were no issues with any of our downstream applications, which included real-time PCR and targeted sequencing. vg ratios vg ratios DN Multi-Sample DN purity ratios average of 3 donors cancer sample mirvana Total RN Isolation Kit PE DN/RN PE DN/RN cancer sample DN Multi-Sample cancer sample RN purity ratios average of 3 donors mirvana Total RN Isolation Kit PE DN/RN PE DN/RN cancer sample 26/28 26/23 26/28 26/23 Figure 2. verage purity ratios of 3 donor samples, obtained using the NanoDrop instrument. () DN and () RN from samples were extracted using the DN Multi-Sample and mirvana Total RN Isolation Kit, respectively; the PE DN/ RN was used to extract both DN and RN from PE samples. The and PE samples were quantified using the Qubit and NanoDrop instruments, respectively.

4 The 21 ioanalyzer system was used to assess RN integrity and size. The RN integrity number (RIN) represents the condition of assayed RN relative to the intact total RN on a scale of 1 1, with 1 indicating completely intact 18S and 28S ribosomal RN (rrn). s expected, with the samples, nice ribosomal RN peaks are present. clearly identifiable small-rn peak is also present since the mirvana kit can isolate small RNs as well. clear distinction between PE and samples is that ribosomal peaks are not usually present in PE samples, leading to a very low RIN, usually <2; however, the size of RN fragments can still be very large. This is observed for both PE Ca and LCa samples processed the percentage of fragments >2 bp was over 6%. With PE samples, RIN numbers hold little value since the samples are inherently degraded, whereas overall fragment sizes play a bigger role (Figure 3). C D Figure 3. RN analysis with the gilent RN 6 Pico kit. RN from () an breast cancer sample and () an lung cancer sample had distinct ribosomal peaks with RIN values of 9 and 8.7, respectively. RN from (C) an PE breast cancer sample and (D) an PE lung cancer sample still had large RN fragments but without clear ribosomal peaks, with RIN values of 1.8 for both.

5 Real-time PCR was performed to assess the functionality of the extracted nucleic acid templates. oth DN and RN from PE samples are fragmented and chemically modified, but the degree of fragmentation and chemical modification vary widely from sample to sample. The fixation process alone does not necessarily lead to fragmentation, but it does in combination with different factors. For example, during the embedding process, the high temperatures required for paraffin infiltration can accelerate chemical reactions that may modify the RN and DN. During storage afterward, these modifications can cause nucleic acid fragmentation and degradation, especially for RN. DN tends not to fragment as easily as RN, so it was not surprising to see equivalent or mostly better C t values for the PE samples compared to the samples (Figure 4), especially for younger blocks. However, since RN is more susceptible to degradation, diminished function as a template for polymerases is expected. We see a difference of.7 cycles in the C t values (Figure 4). Values from older blocks of samples may be more variable, due to a number of factors such as storage conditions or more uncontrolled practices such as fixing the samples for too long. This could lead to more issues with the template being less functional. Nowadays most protocols are more standardized, leading to better handling of the samples. DN and RN libraries were made using 1 ng of template. The number of cycles to be used for the first amplification of target was selected based on the number of primer pairs in the panels and on the sample type ( or PE). asic sequencing run metrics include >93% loading, <% low-quality reads, and % adapter dimer. For DN samples, we tracked the following metrics: mean read lengths, percent mapped, percent reads on target, and percent uniformity. In addition to these basic metrics, we also checked variant calling and compared the results. For mean read lengths the average was >1 bp for either sample type, with very high mapping percentage (Figure ). The data quality was good, with minimum filtering of short reads. The percent reads on target was overall good but lower for the PE samples, which might have been affected by the modified PE DN. On average the percent uniformity was good for both sample types, with values >96%, with the exception of 1 sample at 94%. Figure outlines that the same hotspot mutations were called with similar allele frequencies between and PE samples for both tissue types, allowing the same conclusions to be drawn. DN Multi-Sample kit () PE Ultra kit (PE) mirvana kit () PE Ultra kit (PE) 31 GPDH DN, vs. PE equal mass input GPDH RN, vs. PE equal mass input vg C t 27 2 vg C t a 1b 2a 2b 3a 3b 1a Samples 1b 2a 2b 3a 3b 1a 1b 2a 2b 3a 3b 1a Samples 1b 2a 2b 3a 3b Figure 4. Real-time PCR analysis of DN and RN samples. () Equivalent or mostly better C t values were achieved with the PE DN samples compared to the samples. () difference of.7 cycles in the C t values was observed in the RN samples.

6 For RN samples, the following metrics were tracked: mean read lengths, percent mapped, percent valid reads, and percent on target. lso, we correlated the normalized reads per kilobase million (RPKM) counts for the genes in the RN cancer panel between biological replicates for a given sample and tissue, or between sample types and tissue ( vs. PE). The mean read lengths for these samples were very good, which led to high mapping (>99%) for both sample types, with an average of 127 bp for ; the mean read lengths for the PE samples, as expected, were shorter, with an average of 116 bp, possibly due to the presence of more degradation and fragmentation in the samples. High-quality reads were also obtained, as determined by the percent valid reads with high on-target reads to the genes in the panel. We obtained high correlations to normalized RN counts for all parameters, including between biological sample replicates and, most importantly, between and PE samples for the same tissue type. This gives high confidence that the same genes are being expressed at similar levels (Figure 6). C E Mean read lengths (bp) % Reads on target Sample breast cancer lung cancer DN mean read lengths 1a 1a 1b 2a 2b 3a 3b DN percent reads on target Gene llelic frequency (replicate 1) 1b 2a 2b 3a 3b PE PE llelic frequency (replicate 2) D % Mapped % Uniformity Sample DN percent mapped 1a 1b 2a 2b 3a 3b DN percent uniformity llelic frequency (replicate 1) 1a 1b 2a 2b 3a 3b llelic frequency (replicate 2) PE PE Ref to variant PIK3C Gà KIT PE breast cancer àc TP Tà CTNN PE lung CàG HRS cancer àg Figure. Targeted DN sequencing metrics. () Comparable mean read lengths and () high mapping percentages for both sample types ( and PE breast and lung cancer samples) are shown. (C) On-target percentages were comparable, with better results for samples. On average, 9.% of the sequences were on target for and 91.7% for PE samples. (D) Percent uniformity was also very comparable across the samples types. On average, it was 98.3% for and 97.4% for PE samples. (E) Variant calling the same hotspot mutations were called with similar frequencies.

7 RN mean read lengths RN percent mapped C E Mean read length (bp) % Valid reads (normalized counts) replicate RN percent valid reads breast cancer sample replicates 1a 1b 2a 2b 3a 3b 1a 1b 2a 2b 3a 3b PE replicates PE y =.9729x R 2 =.9934 ( replicates) % Mapped D % Reads on target F (normalized counts) replicate a 1b 2a 2b 3a 3b RN percent reads on target lung cancer sample replicates 1a 1b 2a 2b 3a 3b PE PE y =.9219x R 2 =.9693 replicates ( replicates) G (normalized counts) replicate 1 (normalized counts) replicate 1 PE breast cancer sample replicates H PE lung cancer sample replicates (normalized counts) replicate y = 1.2x R 2 =.9923 PE replicates (PE replicates) (normalized counts) replicate y = 1.411x R 2 = PE replicates (PE replicates) I 1 2 (normalized counts) replicate 1 vs. PE breast cancer sample J 1 2 (normalized counts) replicate 1 vs. PE lung cancer sample (normalized counts) 2 1 y =.94x R 2 =.974 vs. PE ( vs. PE) (normalized counts) 2 1 y =.814x R 2 = vs. PE ( vs. PE) (normalized counts) PE (normalized counts) PE Figure 6. Targeted RN sequencing metrics. () Mean read lengths for PE samples were shorter, as expected, than the samples but still were >11 bp. () Percent mapped was high, yielding comparable results of >99% mapped. (C) Percent valid reads was high, yielding comparable results of >99.% valid reads. (D) Percent reads on target was high, yielding comparable results of >99.% reads on target. (E) Correlation between breast cancer sample replicates; R 2 =.99. (F) Correlation between lung cancer sample replicates; R 2 =.969. (G) Correlation between PE breast cancer sample replicates; R 2 =.992. (H) Correlation between PE lung cancer sample replicates; R 2 =.987. (I) Correlation between and PE breast cancer samples; R 2 =.97. (J) Correlation between and PE lung cancer samples; R 2 =.963.

8 Conclusion Using the workflow described, biomedical researchers can extract RN and DN from and PE samples with sufficient yield, purity, and quality for many downstream genomic and transcriptomic analyses. When researchers aim to obtain both RN and DN from the same sections of PE tissues, high-quality nucleic acids can be obtained with the PE DN/RN. With the samples we outlined here, regardless of the sample-processing techniques either frozen, or fixed and embedded extraction of high-quality nucleic acids was achieved, allowing the same conclusions to be made with confidence. Older sample blocks may be more problematic due to uncontrolled practices, but older PE samples can still be suitable and be used as functional templates for many applications. Ordering information Product Cat. No. Richard-llan Scientific 1% Neutral uffered Formalin 71TS Excelsior ES Tissue Processor 7846 HistoStar Embedding Workstation 811 Richard-llan Scientific Histoplast Paraffin 8332 RNase WY Decontamination Reagent CryoStar NX7 Cryostat 973H HM 3S utomatic Microtome 92 MX3 Premier Disposable Low-Profile Microtome lades or 2. ml microcentrifuge tubes or KingFisher Flex Purification System 463 KingFisher Flex Microtiter Deepwell 96 Plates, sterile 9446 PE DN/RN Qubit 3. Fluorometer Q33216 Qubit dsdn HS ssay Kit Q3284 Qubit RN HS ssay Kit Q328 NanoDrop 2c Spectrophotometer ND2C Find out more at thermofisher.com/ffpeisolation For Research Use Only. Not for use in diagnostic procedures. 217 Thermo Fisher Scientific Inc. ll rights reserved. ll trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. ioanalyzer is a trademark of gilent Technologies, Inc. NanoDrop is a trademark of NanoDrop Technologies. TaqMan is a registered trademark of Roche Molecular Systems, Inc., used under permission and license. COL

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