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. 2013 Jan;59(1):S16-9. doi: 10.1016/j.ymeth.2012.09.011. Epub 2012 Oct 2.


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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Colon / metabolism*
  • Colorectal Neoplasms / metabolism*
  • Cryopreservation*
  • Fixatives
  • Formaldehyde
  • Gene Expression Profiling
  • Humans
  • Paraffin Embedding
  • RNA, Messenger / genetics*
  • RNA, Messenger / isolation & purification
  • RNA, Messenger / metabolism
  • ROC Curve
  • Real-Time Polymerase Chain Reaction


  • Fixatives
  • RNA, Messenger
  • Formaldehyde