Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

The impact of collection and storage of lung tumour tissue on whole genome expression profiling

ABSTRACT: We set out to investigate systematic changes in gene expression that may occur between surgical resection, routine processing in the histopathology department, frozen storage, and long term archiving in FFPE blocks. Lung carcinoma tissue samples were obtained from six patients during tumour resection surgery at the Royal Brompton Hospital, London.. When possible for each patient tissue samples were obtained at five collection points: viable tissue at the time of chest opening (T1a(CO)); immediately after resection (T1b(LR)); after transfer to histopathology (T2a(HP)); after transfer to histopathology and snap freezing (T2b(HP.SF)); and after transfer to histopathology and a week after being snap frozen and archived (T2c(HP.SFA)). FFPE samples of each patientM-^Rs tumour was obtained from histopathology after tissue had been fixed in 10% formalin and then embedded in paraffin blocks according to standard procedure.The T1a(CO), T1b(LR), and T2a(HP) tissues were immediately post sampling placed into RNAlater (Qiagen, USA) for 24 hrs and then frozen at -80oC until RNA extraction. No RNAlater was used for the T2b(HP.SF) and T2c(HP.SFA) samples as these were snap frozen. Total RNA was extracted using the RNeasy Fibrous Tissue Mini Kit (Qiagen, USA) for the majority of time points with the exception of the FFPE samples which were processed using the RecoverAllM-^Y Total Nucleic Acid Isolation Kit for FFPE (Ambion, USA). Illumina Human WG-6 v2 BeadChip microarrays were used to assess global gene expression for each sample.

ORGANISM(S): Homo sapiens

DISEASE(S): squamous cell carcinoma

SUBMITTER: Maxim Freidin

PROVIDER: E-MTAB-581 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Impact of collection and storage of lung tumor tissue on whole genome expression profiling.

Freidin Maxim B MB Bhudia Neesa N Lim Eric E Nicholson Andrew G AG Cookson William O WO Moffatt Miriam F MF

The Journal of molecular diagnostics : JMD 20120110 2

Gene expression profiling could assist in revealing biomarkers of lung cancer prognosis and progression. The handling of biological samples may strongly influence global gene expression, a fact that has not been addressed in many studies. We sought to investigate the changes in gene expression that may occur as a result of sample processing time and conditions. Using Illumina Human WG-6 arrays, we quantified gene expression in lung carcinoma samples from six patients obtained at chest opening be ...[more]

PMID: 22240448

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Project description:In the study of tumor genetics, formalin-fixed paraffin-embedded (FFPE) tumors are the most readily available tissue samples. While DNA derived from FFPE tissue has been validated for array comparative genomic hybridization (aCGH) application, the suitability of such fragmented DNA for single-nucleotide polymorphism (SNP) array analysis has not been well examined. Furthermore, whole-genome amplification (WGA) has been used in the study of small precursor lesions to produce sufficient amount of DNA for aCGH analysis. It is unclear whether the same approach can be extended to SNP analysis. In this study, we examined the utility and limitations of genotyping platform performed on whole-genome amplified DNA from FFPE tumor samples for both copy number and SNP analyses. We analyzed the results obtained using DNA derived from matched FFPE and frozen tissue samples on Affymetrix 250K Nsp SNP array. Two widely used WGA methods, Qiagen (isothermal protocol) and Sigma (thermocycling protocol), were used to determine how WGA methods affect the results. We found that the use of DNA derived from FFPE tumors (without or with WGA) for high-resolution SNP array application can produce a significant amount of false positive and false negative findings. While some of these misinterpretations appear to cluster in genomic regions with high or low GC contents, the majority appears to occur randomly. Only large-scale chromosome LOH (>10Mb) can be reliably detected from FFPE tumor DNA samples (without or with WGA) but not smaller LOH or copy number alterations. Our findings here indicate a need for caution in SNP array data interpretation when using FFPE tumor-derived DNA, particularly with WGA. Affymetrix SNP arrays were performed according to the manufacturer's directions on DNA extracted from cryopreserved and FFPE mesenchymal tumor samples without or with WGA, as well as genomic snap-frozen non-neoplastic tissue DNA from 5 adult individuals to serve as reference DNA. WGA was performed using the REPLI-g® FFPE kit (Qiagen, Valencia, CA, USA) and GenomePlex® Tissue Whole Genome Amplification WGA5 kit (Sigma, Saint Louis, MO, USA) in parallel in accordance with the manufacturers’ protocols. A two- to eight-hour individualized reaction time was used in the Qiagen platform for each sample. A gradient amount of initial DNA (10ng, 30ng, 60ng, 100ng and 150ng) was tested followed by gel electrophoresis and qualitative multiplex PCR assay to determine the quality of post-WGA products. At least four independent experiments were concurrently performed per template amplification. Four separateWGA reaction products were pooled for each sample for subsequent microarray analysis to minimize the amplification bias and allele dropout. One of the Affymetrix GeneChip® Human Mapping 500K Array Set (Nsp 250K SNP array) was used for genotyping analysis. Four gastrointestinal stromal tumors with known cytogenetic aberrations were included. Two cases were sucessfullly amplified and passed the quality tests. A total of 12 samples were compared between each other, including frozen tissue DNA (as reference), frozen tissue DNA with WGA (two platforms), FFPE tissue DNA, and FFPE tissue DNA with WGA (two platforms) from each case.

Dataset Information

The impact of collection and storage of lung tumour tissue on whole genome expression profiling

Publications

Impact of collection and storage of lung tumor tissue on whole genome expression profiling.

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