Optimization of recovery of RNA from formalin-fixed, paraffin-embedded tissue.
Formalin-fixed, paraffin-embedded (FFPE) tissue is the most common specimen available for application of diagnostic assays on tissue after microscopic examination. Not only is there a substantial archive of tissue available, but FFPE tissue remains the best method of preparation for microscopic examination in a routine clinical environment. Molecular assays, especially reverse transcription and polymerase chain reaction and expression array-based assays, offer significant potential as diagnostic, prognostic, and predictive tools, but require high quality RNA. Herein, we have optimized a reliable RNA extraction method for FFPE tissue. It is based on deparaffinization at high temperature coupled with a 3-day lysis at 65 degrees C. The average total RNA yield is 4.5 to 5.5 pg per 1 microm of archival FFPE tissue, and 260/280 ratios are between 1.80 and 1.95. The extracted RNA has a modal fragment length between 100 and 200 nt by the Bioanalyzer analysis. Although modal lengths of RNA fragments were shorter, reverse transcription and polymerase chain reaction was able to amplify amplicons in range of 300 bp. Pretreatment with RNA, later followed by formalin fixation, did not result in improving the RNA quality, but did improve RNA yield. Our method improves the utility of FFPE tissue for molecular profiling studies.
Chung
Joon-Yong
JY
Tissue Array Research Program, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4605, USA.
Braunschweig
Till
T
Hewitt
Stephen M
SM
D000818:Animals; D002021:Buffers; D015925:Cryopreservation; D005404:Fixatives; D005557:Formaldehyde; D006801:Humans; D007668:Kidney / Q000737:chemistry; D008297:Male; D016612:Paraffin Embedding; D012313:RNA / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D020871:RNA Stability; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013696:Temperature; D013997:Time Factors; D016707:Tissue Fixation
D002021:Buffers; D005404:Fixatives; D005557:Formaldehyde; D012313:RNA