Whole-genome gene expression profiling of formalin-fixed, paraffin-embedded tissue samples.
BACKGROUND
We have developed a gene expression assay (Whole-Genome DASL), capable of generating whole-genome gene expression profiles from degraded samples such as formalin-fixed, paraffin-embedded (FFPE) specimens.


RESULTS
We demonstrated a similar level of sensitivity in gene detection between matched fresh-frozen (FF) and FFPE samples, with the number and overlap of probes detected in the FFPE samples being approximately 88% and 95% of that in the corresponding FF samples, respectively; 74% of the differentially expressed probes overlapped between the FF and FFPE pairs. The WG-DASL assay is also able to detect 1.3-1.5 and 1.5-2 -fold changes in intact and FFPE samples, respectively. The dynamic range for the assay is approximately 3 logs. Comparing the WG-DASL assay with an in vitro transcription-based labeling method yielded fold-change correlations of R(2) approximately 0.83, while fold-change comparisons with quantitative RT-PCR assays yielded R(2) approximately 0.86 and R(2) approximately 0.55 for intact and FFPE samples, respectively. Additionally, the WG-DASL assay yielded high self-correlations (R(2)>0.98) with low intact RNA inputs ranging from 1 ng to 100 ng; reproducible expression profiles were also obtained with 250 pg total RNA (R(2) approximately 0.92), with approximately 71% of the probes detected in 100 ng total RNA also detected at the 250 pg level. When FFPE samples were assayed, 1 ng total RNA yielded self-correlations of R(2) approximately 0.80, while still maintaining a correlation of R(2) approximately 0.75 with standard FFPE inputs (200 ng).


CONCLUSIONS
Taken together, these results show that WG-DASL assay provides a reliable platform for genome-wide expression profiling in archived materials. It also possesses utility within clinical settings where only limited quantities of samples may be available (e.g. microdissected material) or when minimally invasive procedures are performed (e.g. biopsied specimens).
April
Craig
C
Illumina, Inc., San Diego, California, United States of America.
Klotzle
Brandy
B
Royce
Thomas
T
Wickham-Garcia
Eliza
E
Boyaniwsky
Tanya
T
Izzo
John
J
Cox
Donald
D
Jones
Wendell
W
Rubio
Renee
R
Holton
Kristina
K
Matulonis
Ursula
U
Quackenbush
John
J
Fan
Jian-Bing
JB
D045744:Cell Line, Tumor; D005557:Formaldehyde / Q000737:chemistry*; D005629:Frozen Sections; D020869:Gene Expression Profiling / Q000379:methods*; D015972:Gene Expression Regulation, Neoplastic; D015894:Genome, Human / Q000235:genetics*; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D016612:Paraffin Embedding / Q000379:methods*; D020871:RNA Stability / Q000235:genetics; D012334:RNA, Neoplasm / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016707:Tissue Fixation / Q000379:methods*; D014158:Transcription, Genetic
D012334:RNA, Neoplasm; D005557:Formaldehyde