The challenge of gene expression profiling in heterogeneous clinical samples

Rodríguez-González, F. Germán; Mustafa, Dana A. M.; Mostert, Bianca; Sieuwerts, Anieta M.

doi:10.1016/j.ymeth.2012.05.005

Cited by 18 publications

(10 citation statements)

References 148 publications

(141 reference statements)

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“…At P <0.01 the correlation between the pairs ranged between 0.74–0.99 and the cluster analysis showed a match of 25 out of the 27 pairs. The 2 non-matching samples in this analysis clustered to another breast cancer sample, which may be explained by the heterogeneity within the tissue [ 10 , 19 , 24 ]. The number of paired samples clustering together decreased after increasing the number of probes included in the analysis.…”

Section: Discussionmentioning

confidence: 99%

A Method to Correlate mRNA Expression Datasets Obtained from Fresh Frozen and Formalin-Fixed, Paraffin-Embedded Tissue Samples: A Matter of Thresholds

et al. 2015

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BackgroundGene expression profiling of tumors is a successful tool for the discovery of new cancer biomarkers and potential targets for the development of new therapeutic strategies. Reliable profiling is preferably performed on fresh frozen (FF) tissues in which the quality of nucleic acids is better preserved than in formalin-fixed paraffin-embedded (FFPE) material. However, since snap-freezing of biopsy materials is often not part of daily routine in pathology laboratories, one may have to rely on archival FFPE material. Procedures to retrieve the RNAs from FFPE materials have been developed and therefore, datasets obtained from FFPE and FF materials need to be made compatible to ensure reliable comparisons are possible.AimTo develop an efficient method to compare gene expression profiles obtained from FFPE and FF samples using the same platform.MethodsTwenty-six FFPE-FF sample pairs of the same tumors representing various cancer types, and two FFPE-FF sample pairs of breast cancer cell lines, were included. Total RNA was extracted and gene expression profiling was carried out using Illumina’s Whole-Genome cDNA-mediated Annealing, Selection, extension and Ligation (WG-DASL) V3 arrays, enabling the simultaneous detection of 24,526 mRNA transcripts. A sample exclusion criterion was created based on the expression of 11 stably expressed reference genes. Pearson correlation at the probe level was calculated for paired FFPE-FF, and three cut-off values were chosen. Spearman correlation coefficients between the matched FFPE and FF samples were calculated for three probe lists with varying levels of significance and compared to the correlation based on all measured probes. Unsupervised hierarchical cluster analysis was performed to verify performance of the included probe lists to compare matched FPPE-FF samples.ResultsTwenty-seven FFPE-FF pairs passed the sample exclusion criterion. From the profiles of 27 FFPE and FF matched samples, the best correlating probes were identified for various levels of significance (Pearson P<0.01, n = 1,432; P<0.05, n = 2,530; and P<0.10, n = 3,351 probes). Unsupervised hierarchical clustering of the 27 pairs using the resulting probes yielded 25, 21, and 19 correctly clustered pairs, respectively, compared to 1 pair when all probes were used.ConclusionThe proposed method enables comparison of gene expression profiles of FFPE and/or FF origin measured on the same platform.

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Section: Discussionmentioning

confidence: 99%

A Method to Correlate mRNA Expression Datasets Obtained from Fresh Frozen and Formalin-Fixed, Paraffin-Embedded Tissue Samples: A Matter of Thresholds

et al. 2015

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“…Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque density gradient centrifugation at 400 g for 40 min at room temperature. The cells at the interface were washed twice with phosphate buffered saline (PBS) [27, 28]. …”

Section: Methodsmentioning

confidence: 99%

MiRNA and mRNA Profiling in Systemic Lupus Reveals a Novel Set of Cytokine - Related miRNAs and their Target Genes in Cases With and Without Renal Involvement

Ghods

Sarikaya

Arda

et al. 2017

Kidney Blood Press Res

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Background/Aims: MiRNAs transpire as promising elements in molecular medicine for the identification of new diagnostic, prognostic and targeting therapeutic biomarkers. This study consisted of four steps: First, to investigate one or a group of specific diagnostic miRNAs for Systemic Lupus Erythematosus (SLE) disease in patients with and without renal involvement, second, to identify cytokines genes’ expression profiling, third, comparing the profiles with related amounts in the serum and finally, to study target-gene-mediated functional roles of miRNAs, which have been correlated to disease development and progression. Methods: In order to use in microarray assays total RNA and miRNAs were isolated from blood and serum samples that were obtained from 16 SLE patients (9 with renal involvement and 7 without renal involvement). Taking coexistence of factors such as hypocomplementemia, positive ANA and anti-DNA into account, obtained data were processed. For each differentially expressed miRNA, potential target genes were predicted by microRNAorg, TargetScan and PITA prediction tools. Obtained mRNA profiling data were interrogated for the target genes. MiRNA and mRNA microarray results were confirmed by QRT-PCR. Finally, the amounts of cytokines were measured by multiplex ELISA method. Results: The results of study showed that among differentially expressed miRNAs in SLE patients with renal involvement compared to those without renal involvement, hsa-miR-766-3p, may play pivotal roles in PI3K-AKT-mTOR pathway. In addition according to the obtained data it is suggested that blood-borne proinflammatory cytokines such as IL-4, IL-6 and TNF-α alongside with disease stage and severity may contribute to this differential expression of these miRNA which may be leading to insulin resistance. Finally, hsa-miR-621, which was differentially expressed in hypertensive SLE patients without renal involvement and a positive ANA test with its predicted target gene “Kallikrein-related peptidase 9” may play a role in the pathophysiology of hypertension in SLE. Conclusions: We reported some human miRNAs which were differentially expressed in SLE patients according to disease activity and renal involvement. Larger studies are necessary to confirm our findings and detect further biomarkers.

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“…These techniques can enhance the quantification of specific DNA sequences, with the corresponding analytical results being applicable to disease diagnosis (e.g., influenza) [5][6][7][8], environmental monitoring, and microbiological food analysis [9][10][11]. In the case of qPCR, the target gene fragment is monitored by fluorescence spectroscopy, allowing the quantitative analysis of amplified small DNAs.…”

Section: Introductionmentioning

confidence: 99%

Separation of Small DNAs by Gel Electrophoresis in a Fused Silica Capillary Coated with a Negatively Charged Copolymer

Nakazumi

Hara

2017

Separations

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Active development of compact analytical instruments suitable for point-of-care testing (POCT) requires optimization of existing methods. To aid the development of capillary gel electrophoresis instruments for POCT, we attempted to separate polymerase chain reaction products (small DNAs) using a short, fused silica capillary coated with an acrylamide (AM)/acrylic acid (AA) copolymer (poly(AM-co-AA)). To realize the high capability of this capillary to separate small DNAs, the magnitude of electroosmotic flow (EOF) was controlled by varying the content of negatively charged AA in the copolymer, which significantly affected the separation ability. At an AA content ≥3.75 mol %, sample DNAs could not be injected into the copolymer-coated capillary owing to strong EOF, whereas a 100 bp DNA ladder sample was successfully separated at an AA content of ≤3.5 mol %, showing that even slight AA content variations impact DNA flow. EOF values measured using a neutral coumarin 334 solution suddenly decreased at an AA content of 3.5 mol % relative to those at an AA content of ≥3.75 mol %. Theoretical plate values revealed that an AA content of 2.75 mol % was optimal for separating ladder DNAs with sizes ≥600 bp. Hence, EOF control achieved by varying the amount of negatively charged AA in the poly(AM-co-AA) coating can promote further development of short capillaries for POCT applications.

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The challenge of gene expression profiling in heterogeneous clinical samples

Cited by 18 publications

References 148 publications

A Method to Correlate mRNA Expression Datasets Obtained from Fresh Frozen and Formalin-Fixed, Paraffin-Embedded Tissue Samples: A Matter of Thresholds

A Method to Correlate mRNA Expression Datasets Obtained from Fresh Frozen and Formalin-Fixed, Paraffin-Embedded Tissue Samples: A Matter of Thresholds

MiRNA and mRNA Profiling in Systemic Lupus Reveals a Novel Set of Cytokine - Related miRNAs and their Target Genes in Cases With and Without Renal Involvement

Separation of Small DNAs by Gel Electrophoresis in a Fused Silica Capillary Coated with a Negatively Charged Copolymer

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