Gene Expression Analysis Comparing Two RNA .Gene Expression Analysis Comparing Two RNA Isolation

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Transcript of Gene Expression Analysis Comparing Two RNA .Gene Expression Analysis Comparing Two RNA Isolation

  • High Pure FFPET Application Note 1

    Gene Expression Analysis Comparing Two RNA Isolation Methods from Recent and Older Archived Formalin-Fixed, Paraffin-Embedded Tissue (FFPET) Samples

    AbstractFormalin-fixed, paraffin-embedded tissue samples are widely available for retrospective studies. However the quantity and the quality of the isolated RNA can limit downstream applications. Our aims were to compare the High Pure FFPET RNA Isolation Kit with a commercially available FFPE RNA isolation kit with regard to the quality and quantity of the isolated RNA.

    RNA isolation was performed using High Pure FFPET RNA Isolation Kit and RNeasy FFPE Kit (Qiagen), from slides cut from FFPE blocks containing human colorectal cancer (CRC) and normal adjacent colon tissue (NAT) with different storage time, archived samples older than 5 years and recent samples younger than 6 months. After quantity and quality assessment, gene expression analysis of three colorectal cancer markers was performed using RT-qPCR to evaluate the performance of the isolated RNA samples.

    The High Pure FFPET RNA Isolation Kit purification resulted in higher yield and RIN values compared to the RNeasy FFPE Kit from both archived and recent FFPE samples. Samples isolated using the High Pure FFPET RNA Isolation Kit are highly suited for comparison of marker gene expression levels. In all settings (archived/recent tissue, normal/tumor tissue, all marker genes), earlier absolute Cq values were achieved with High Pure FFPET RNA Isolation Kit, indicating higher sensitivity and amplificability. Differences in marker gene expression levels are clearly detectable between normal and tumor tissue. Relative expression of marker genes is lower in normal tissue compared to tumor tissue.

    In conclusion, high quality and quantity of total RNA can be isolated from FFPE blocks using High Pure FFPET RNA Isolation Kit, providing reliable starting material for gene expression studies.

    For life science research only. Not for use in diagnostic procedures.

    October 2012

    Alexandra Kalmar*, Kinga Tth, Katalin Leiszter, rpd V. Patai, Andrea Schller 2nd Department of Internal Medicine, Semmelweis University, Budapest, Hungary

    Barnabs Wichmann*, Orsolya Galamb, Sndor Spisk, Zsolt Tulassay, Bla Molnr Molecular Medicine Research Unit, Hungarian Academy of Sciences, Budapest, Hungary

    *These authors contributed equally to this work

  • Formalin-fixed, paraffin-embedded (FFPE) tissue is the most widely available sample type. This makes it a valuable starting material for retrospective studies. However, the applicability of this type of sample in gene expression studies is limited due to the low yield and purity of the isolated RNA samples. In addition to tissue fixation, subsequent storage time of the tissue blocks is one of the main factors that can influence the quality and quantity of the isolated nucleic acids, as long-term storage can result in lower yield [1].

    Introduction

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    Materials and Methods

    Sample material and sample collectionFormalin-fixed, paraffin-embedded tissue blocks were prepared from surgically removed colorectal cancer (CRC, Dukes B-C), and from the belonging normal adjacent (NAT) tissue specimens (referred to as "normal" in the text).

    Two groups of FFPE blocks were analyzed with different storage time, archived samples older than 5 years (n = 24; 12 CRC, 12 NAT), and recent samples younger than 6 months (n = 24; 12 CRC, 12 NAT). Written informed consent was provided by all patients. The study was approved by the local ethics committee.

    RNA isolation10 m thick sections were cut from formalin-fixed, paraffin-embedded blocks. Each section was transferred into micro-centrifuge tubes. Before both isolations, deparaffinization was performed with 1 ml xylene with 10 minutes incubation time twice, and 1 ml absolute ethanol for 10 minutes twice. Total RNA isolation was performed from each air dried

    Quantitative and qualitative analysis of the isolated RNA samples The concentration of the isolated RNA was measured using the NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific Inc., USA, Waltham). RNA quality was assessed using the RNA Integrity Number (RIN) measured by RNA 6000 Pico LabChip kit on a microcapillary electrophoresis system (Agilent BioAnalyzer 2100).

    deparaffinized section with High Pure FFPET RNA Isolation Kit (Roche Applied Science, Penzberg, Germany) and RNeasy FFPE Kit (QIAGEN GmbH, Hilden, Germany) in duplicates, according to the manufacturers instructions. Both protocols include DNase treatment. To avoid any sample bias, sequential sections were used for RNA isolation with High Pure FFPET RNA Isolation Kit and RNeasy FFPE Kit.

    To analyze possible contamination of genomic DNA in the isolated RNA samples, RT-qPCR reactions were performed using a cyclophilin A (CycA) assay with 4 normal and 4 CRC total RNA samples isolated with both methods. Dilution series of human genomic DNA (20 ng, 2 ng, 0.2 ng, 0.02 ng, 0.002 ng final concentrations) were prepared as standards. After denaturation at +95 C for 10 min, 45 cycles of ampli-fication were performed: +95 C for 5 sec, +55 C for 10 sec and signal detection at +72 C for 18 sec, and cooling at +40 C for 30 sec.

    Downstream applications require high quality RNA, which is largely influenced by the isolation procedure [2]. Here we present a comparative study using two commercially available kits for RNA isolation from FFPE tissue using older archived and recent tissue samples. For testing the downstream appli-cability of the isolated samples, gene expression of three recently identified colorectal cancer-specific discriminatory transcripts [3] was determined using real-time PCR.

  • Materials and Methods

    3

    Table 1: Analyzed markers with amplicon lengths.

    Real-time PCR analysisUsing the Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics), 250 ng of total RNA were reverse transcribed with a combination of anchored-oligo(dT) and random hexamer primers. Gene expression analysis was performed for three colorectal cancer specific markers [3] (COL12A1, CXCL2, CHI3L1), and 18S ribosomal RNA as housekeeping gene. RealTime ready assays from Universal Probe Library (Roche Applied Science) were used with forward and reverse primers (400 nM), and fluorescently labeled hydrolysis probes (200 nM) lyophilized into 384 well plates (Table 1).

    Statistical analysis of OD ratiosAnalytical OD ratio data of the RNA eluates were represen-ted in boxplots, with red dots for each individual value. Boxplots indicate five number summaries of data such as sample minimum, lower quartile (Q1), median (Q2), upper quartile (Q3) and sample maximum. It also displays the full range of variation and likely range of variation (IQR). This later is the difference of Q1 and Q3. Outliers are data points

    Gene symbol Gene name Amplicon length RealTime ready Assay ID

    CHI3L1 chitinase 3-like 1 76 103035

    COL12A1 collagen, type XII, alpha 1 66 103045

    CXCL2 chemokine (C-X-C motif) ligand 2 95 103070

    RN18S1 RNA, 18S ribosomal 1, 18S ribosomal RNA 73 104092

    Real-time PCR reactions were performed in a final volume of 10 l using 5 ng cDNA/well and 5 l LightCycler 480 Probes Master. The epMotion 5070 liquid handling robot (Eppendorf, Hamburg, Germany) was used for pipetting. Thermal cycling conditions on the LightCycler 480 System were the following: Enzyme activation: +95 C for 10 min, 45 cycles of amplification: +95 C for 10 sec, +60 C for 30 sec, and signal detection at +72 C for 1 sec, and cooling at +40 C for 30 sec.

    which are either 3xIQR or more above Q3 or 3xIQR or more below Q1. Box represents 50% of data, whiskers represent 24.65 24.65% of data either above or below the boxplot. Data points which are beyond each quartile with 1.5 IQR are deemed as potential outliers. Gene expression analysis was performed with relative quantification using the ddCq method with 18S ribosomal RNA housekeeping gene.

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    Quantity and quality of the isolated RNA samplesTotal RNA was isolated from 12 archived and 12 recent CRC and normal FFPE blocks. Isolations were performed in duplicates. Altogether 96 isolations were performed with

    RNA yield by OD260The High Pure FFPET RNA Isolation Kit resulted in significantly higher yield (archived tissue: 6.96 3.9 g RNA/slide; recent tissue: 7.67 4.8 g RNA/slide) compared to the RNeasy FFPE Kit (archived tissue 2.27 1.57 g RNA/slide; recent tissue: 2.95 2.34 g RNA/slide) (Table 2). Yield was consistently higher using the High Pure Kit (Figure 1).

    Results and Discussion

    4

    Table 2. Comparison of the two isolation methods from older archived and recent FFPE tissue samples. Mean values standard deviation are shown.

    Figure 1. Comparison of RNA yields from individual sequential FFPE sections. RNA yields are presented for each of the 24 replicates, resulting in mean yields in Table 2. As expected, RNA yields vary between sections. The High Pure FFPET RNA Isolation Kit consistently generated higher yields compared to RNeasy FFPE Kit.

    High Pure FFPET RNA Isolation Kit and 96 with Qiagen's RNeasy FFPE Kit. Comparison of the RNA quantity and quality results are summarized in Table 2.

    Archived FFPE tissue Recent FFPE tissue

    High Pure Kit RNeasy FFPE Kit P value High Pure Kit RNeasy FFPE Kit P value

    Normal samples

    Yield (g) 4.19 2.45 1.65 1.4 1.41 10-5 5.69 5.09 3.45 4.17 0.08

    OD260/280 1.86 0.01 1.90 0.08 0.13 1.92 0.05 1.94 0.07 0.34

    OD260/230 1.69 0.35 1.82 0.33 0.14 1.83 0.23 1.75 0.29 0.27

    RIN 2.36 0.32 1.97 0.5 0.08 2.85 0.21 2.70 1.05 0.85

    Tumor samples

    Yield (g) 9.72 3.03 2.59 1.4 8.23 10-16 9.65 3.59 3.97