Reliable gene expression measurements from fine needle aspirates of pancreatic tumors: effect of amplicon length and quality assessment.
OBJECTIVE
Biomarker use for pancreatic cancer diagnosis has been impaired by a lack of samples suitable for reliable quantitative RT-PCR (qRT-PCR). Fine needle aspirates (FNAs) from pancreatic masses were studied to define potential causes of RNA degradation and develop methods for accurately measuring gene expression.


METHODS
Samples from 32 patients were studied. RNA degradation was assessed by using a multiplex PCR assay for varying lengths of glyceraldehyde-3-phosphate dehydrogenase, and effects on qRT-PCR were determined by using a 150-bp and a 80-bp amplicon for RPS6. Potential causes of and methods to circumvent RNA degradation were studied by using FNAs from a pancreatic cancer xenograft.


RESULTS
RNA extracted from pancreatic mass FNAs was extensively degraded. Fragmentation was related to needle bore diameter and could not be overcome by alterations in aspiration technique. Multiplex PCR for glyceraldehyde-3-phosphate dehydrogenase could distinguish samples that were suitable for qRT-PCR. The use of short PCR amplicons (<100 bp) provided reliable gene expression analysis from FNAs. When appropriate samples were used, the assay was highly reproducible for gene copy number with minimal (0.0003 or about 0.7% of total) variance.


CONCLUSIONS
The degraded properties of endoscopic FNAs markedly affect the accuracy of gene expression measurements. Our novel approach to designate specimens "informative" for qRT-PCR allowed accurate molecular assessment for the diagnosis of pancreatic diseases.
Anderson
Michelle A
MA
Department of Internal Medicine, Division of Gastroenterology, University of Michigan Health System, Ann Arbor, Michigan 48109-0362, USA. maaander@umich.edu
Brenner
Dean E
DE
Scheiman
James M
JM
Simeone
Diane M
DM
Singh
Nalina
N
Sikora
Matthew J
MJ
Zhao
Lili
L
Mertens
Amy N
AN
Rae
James M
JM
D000818:Animals; D001483:Base Sequence; D001707:Biopsy, Needle; D017931:DNA Primers; D015870:Gene Expression; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D010190:Pancreatic Neoplasms / Q000201:enzymology / Q000235:genetics* / Q000473:pathology; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases