Dataset Information

Reliable gene expression measurements from fine needle aspirates of pancreatic tumors: effect of amplicon length and quality assessment.

ABSTRACT:

Background and aims

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.

SUBMITTER: Anderson MA

PROVIDER: S-EPMC2928420 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Reliable gene expression measurements from fine needle aspirates of pancreatic tumors: effect of amplicon length and quality assessment.

Anderson Michelle A MA 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

The Journal of molecular diagnostics : JMD 20100813 5

<h4>Background and aims</h4>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.<h4>Methods</h4>Samples from 32 patients were studied. RNA degradation was assessed by using a multiplex PCR assay for varying lengths of glyceraldehyde-3-phospha ...[more]

PMID: 20709792

Similar Datasets

Project description:Background & aimsFinite duration of treatment associated with HBsAg loss is the current goal for improved therapeutic approaches against chronic HBV infection, as it indicates elimination or durable inactivation of intrahepatic covalently closed circular DNA (cccDNA). To assist drug development, the definition of early predictive markers of HBsAg loss by assessing their value in reflecting intrahepatic cccDNA levels and transcriptional activity is essential. Fine needle aspirates (FNAs) have recently emerged as a less invasive alternative to core liver biopsy (CLB) and showed to be useful for investigating intrahepatic immune responses. The aim of this study was to optimise and validate the use of FNA vs. CLB to evaluate the intrahepatic viral reservoir.MethodsPaired FNA/CLB samples were obtained from patients with HBeAg+ chronic hepatitis (n = 4), HBeAg- chronic hepatitis (n = 4), and HBeAg- chronic infection (n = 1). One HBeAg+ patient was undergoing tenofovir treatment. HBV 3.5-kb RNA and cccDNA were quantified by droplet digital PCR.ResultscccDNA was quantifiable in all but one FNA/CLB pair, showing the highest levels in untreated HBeAg+ patients, except for the tenofovir-treated patient. Similarly, 3.5-kb RNA was detectable in all but one FNA sample and showed higher levels in HBeAg+ patients. When comparing cccDNA and 3.5-kb RNA quantification in FNA vs. CLB samples, no statistically significant differences were identified.ConclusionsThese results demonstrate the possibility to quantify cccDNA and assess its transcriptional activity in patients with chronic hepatitis B by combining FNA and droplet digital PCR. This supports the use of FNA in clinical trials to evaluate the intrahepatic viral reservoir during the development of new antivirals and immunomodulatory agents.Impact and implicationsChronic hepatitis B infection is characterised by a complex interplay between immune responses and viral replication in the liver, which determines the long-term outcome of the disease. In this study, we show that fine needle aspiration of the liver, a less-invasive alternative to core biopsies, allows the assessment of the hepatic viral reservoir.

Dataset Information

Reliable gene expression measurements from fine needle aspirates of pancreatic tumors: effect of amplicon length and quality assessment.

Background and aims

Methods

Results

Conclusions

Publications

Reliable gene expression measurements from fine needle aspirates of pancreatic tumors: effect of amplicon length and quality assessment.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets