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Gene expression analysis of normal and colorectal cancer tissue samples from fresh frozen and matched formalin-fixed, paraffin-embedded (FFPE) specimens after manual and automated RNA isolation

Kalmár, Alexandra and Wichmann, Barnabás and Galamb, Orsolya and Spisák, Sándor and Tóth, Kinga and Leiszter, Katalin and Tulassay, Zsolt and Molnár, Béla (2013) Gene expression analysis of normal and colorectal cancer tissue samples from fresh frozen and matched formalin-fixed, paraffin-embedded (FFPE) specimens after manual and automated RNA isolation. METHODS-A COMPANION TO METHODS IN ENZYMOLOGY, 59 (1). S16-S19. ISSN 1046-2023

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Abstract

Although RNA isolation is a routine process in gene expression analysis studies, the applicability of most widely available formalin-fixed, paraffin-embedded (FFPE) samples is still limited compared to fresh frozen tissue samples due to the lower quality of the isolated RNA. Recently, novel automated isolation methods were developed in order to reduce manual sample handling and increase RNA quality and quantity. Here we present a comparison of the performance of fresh frozen and matched FFPE tissue samples obtained from the same surgically removed colonic specimens (10 normal, 10 CRC) in RT-PCR experiments. RNA isolations were performed with the automated MagNA Pure 96 Cellular RNA Large Volume Kit (Roche) compared to the manual RNeasy FFPE Mini Kit (Qiagen). Gene expression analysis of a colorectal cancer-specific marker set (with 7 genes: COL12A1, CXCL1, CXCL2, GREM1, IL1B, IL8, SLC7A5) was performed with array real-time PCR using Transcriptor First Strand cDNA Synthesis Kit (Roche) and RealTime ready assays on LightCycler® 480 System (Roche). On the basis of the gene expression of the analyzed markers, fresh frozen tumorous and normal samples could be distinguished with 100% sensitivity and 100% specificity after both isolation methods. The FFPE samples could be distinguished by similarly high specificity and sensitivity with the MagNA Pure 96 isolated samples (sensitivity: 90,0%; specificity: 90,0%) and the samples isolated with manual Qiagen method (sensitivity: 85,0%; specificity: 70,0%). According to these results, FFPE samples isolated by automated methods can serve as valuable source for retrospective gene expression studies in the field of biomarker discovery and development. © 2012 Elsevier Inc.

Item Type: Article
Additional Information: A Scopus-ban 179-182. oldalszámozás szerepel, a fulltexten és a többi adatbázisban az S16-S19.
Uncontrolled Keywords: Sensitivity and Specificity; review; reverse transcription polymerase chain reaction; real time polymerase chain reaction; priority journal; PERFORMANCE; human tissue; human; general medical device; Gene Expression; cancer tissue; unclassified drug; SLC7A5 protein; PROTEIN; paraffin; interleukin 8; interleukin 1beta; GREM1 protein; fresh frozen plasma; CXCL2 chemokine; CXCL1 chemokine; complementary dna; COL12A1 protein; RNA isolation; Quantitative real-time PCR; Paraffin-embedded tissue; MagNA Pure 96 Instrument; Fresh frozen tissue; Formalin-fixed; colorectal cancer
Subjects: Q Science / természettudomány > QH Natural history / természetrajz > QH301 Biology / biológia > QH3011 Biochemistry / biokémia
Q Science / természettudomány > QH Natural history / természetrajz > QH301 Biology / biológia > QH3015 Molecular biology / molekuláris biológia
SWORD Depositor: MTMT SWORD
Depositing User: MTMT SWORD
Date Deposited: 06 Feb 2014 03:18
Last Modified: 06 Feb 2014 03:18
URI: http://real.mtak.hu/id/eprint/9892

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