Enhanced stability of microRNA expression facilitates classification of FFPE tumour samples exhibiting near total mRNA degradation.
BACKGROUND
As degradation of formalin-fixed paraffin-embedded (FFPE) samples limits the ability to profile mRNA expression, we explored factors predicting the success of mRNA expression profiling of FFPE material and investigated an approach to overcome the limitation.


METHODS
Bladder (n=140, stored 3-8 years) and cervix (n=160, stored 8-23 years) carcinoma FFPE samples were hybridised to Affymetrix Exon 1.0ST arrays. Percentage detection above background (%DABG) measured technical success. Biological signal was assessed by distinguishing cervix squamous cell carcinoma (SCC) and adenocarcinoma (AC) using a gene signature. As miR-205 had been identified as a marker of SCC, precursor mir-205 was measured by Exon array and mature miR-205 by qRT-PCR. Genome-wide microRNA (miRNA) expression (Affymetrix miRNA v2.0 arrays) was compared in eight newer FFPE samples with biological signal and eight older samples without.


RESULTS
RNA quality controls (QCs) (e.g., RNA integrity (RIN) number) failed to predict profiling success, but sample age correlated with %DABG in bladder (R=-0.30, P<0.01) and cervix (R=-0.69, P<0.01). Biological signal was lost in older samples and neither a signature nor precursor mir-205 separated samples by histology. miR-205 qRT-PCR discriminated SCC from AC, validated by miRNA profiling (26-fold higher in SCC; P=1.10 × 10(-5)). Genome-wide miRNA (R=0.95) and small nucleolar RNA (R=0.97) expression correlated well in the eight newer vs older FFPE samples and better than mRNA expression (R=0.72).


CONCLUSIONS
Sample age is the best predictor of successful mRNA profiling of FFPE material, and miRNA profiling overcomes the limitation of age and copes well with older samples.
Translational Radiobiology Group, School of Cancer and Enabling Sciences, Manchester Academic Health Science Centre, The University of Manchester, Christie Hospital NHS Trust, Wilmslow Road, Manchester M20 4BX, UK.
J S
JS
Hall
J
J
Taylor
H R
HR
Valentine
J J
JJ
Irlam
A
A
Eustace
P J
PJ
Hoskin
C J
CJ
Miller
C M L
CM
West
D000230:Adenocarcinoma / Q000235:genetics; D002294:Carcinoma, Squamous Cell / Q000235:genetics; D005260:Female; D005404:Fixatives / Q000494:pharmacology; D005557:Formaldehyde / Q000494:pharmacology; D020869:Gene Expression Profiling / Q000379:methods*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008297:Male; D035683:MicroRNAs / Q000378:metabolism*; D016612:Paraffin Embedding / Q000379:methods*; D020871:RNA Stability; D012333:RNA, Messenger / Q000378:metabolism*; D013997:Time Factors; D014021:Tissue Preservation; D001749:Urinary Bladder Neoplasms / Q000235:genetics*; D002583:Uterine Cervical Neoplasms / Q000235:genetics*
D005404:Fixatives; D005557:Formaldehyde; D035683:MicroRNAs; D012333:RNA, Messenger; C534425:MIR205, human