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| Status |
Public on Oct 10, 2015 |
| Title |
Optimizing microarray gene expression profiling workflow for formalin-fixed paraffin-embedded tissues |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by array
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| Summary |
The use of nucleic acids from formalin-fixed paraffin-embedded (FFPE) tissues for high-throughput molecular techniques, such as microarray gene expression profiling has become widespread in molecular research area. However, working with FFPE tissues is challenging because of degradation, cross-linking with proteins, and RNA chemical modifications. Also, there is no generally accepted procedure for RNA extraction to microarray analysis. Thus, there is a need for a standardized workflow for FFPE samples to study microarray transcriptome profiling. Therefore, the main purpose of this study was to conduct a standardized process from deparaffinization to RNA extraction and microarray gene expression analysis. Firstly, deparaffinization procedure was optimized for FFPE samples and then Trizol, PicoPure RNA isolation kit, and Qiagen RNeasy FFPE kit performances were compared in terms of yield and purity. Finally, two different cRNA/cDNA preparation and labeling protocols with two different array platforms (Affymetrix Human Genome U133 Plus 2.0 and U133_X3P) were also evaluated to determine which combination gives the best percentage of present call. Our optimization study shows that the Qiagen RNeasy FFPE kit with modified deparaffinization step gives better results (RNA quantity and quality) than the other two isolation kits. The Ribo-SPIA protocol and U133_X3P array combination gave a significantly higher percentage of present calls than the 3’ IVT cDNA amplification and labeling system. However, no significant differences were found between the two array platforms. These results present a workflow for microarray gene expression profiling of FFPE tissues. The findings also indicate that sufficient quality gene expression data can be obtained from FFPE-derived RNA.
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| Overall design |
Four FFPE colon cancer tissue sections were used for this study. 4 sections (each 8 μm thick) were cut from each sample and used for RNA extractions. RNA amplifications were performed using the Affymetrix 3’ IVT Kit and the NuGEN Ovation FFPE WTA system. Four different hybridization combinations were performed on four labeled samples which were amplified by two different kits. Human Genome U133 Plus 2.0 and U133_X3P arrays were used for these four combinations. Three of these four samples have matched control samples. In this study gene expression data analysis was also performed to detect differentially expressed genes for these three matched samples. These three matched normal controls were independently isolated, amplified, fragmented and labeled for another larger microarray gene expression study using our optimized workflow. These samples were hybridized to same microarray platform (U133_X3P Array) as the matched tumor samples.
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| Contributor(s) |
Belder N, Coşkun Ö, Erdoğan BD, Dağ Öi, Savaş B, Ensari A, Özdağ H |
| Citation(s) |
26981433 |
| Submission date |
Oct 09, 2015 |
| Last update date |
Mar 25, 2019 |
| Contact name |
Nevin Belder |
| E-mail(s) |
nevinbelder@gmail.com
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| Organization name |
Ankara University
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| Department |
Biotechnology
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| Lab |
Genomics
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| Street address |
De gol
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| City |
Ankara |
| State/province |
Tandogan |
| ZIP/Postal code |
06100 |
| Country |
Turkey |
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| Platforms (2) |
| GPL570 |
[HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array |
| GPL1352 |
[U133_X3P] Affymetrix Human X3P Array |
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| Samples (19)
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| GSM1904888 |
Tumor_08/40_HGU133 Plus 2.0+3' IVT kit |
| GSM1904889 |
Tumor_08/95_HGU133 Plus 2.0+3' IVT kit |
| GSM1904890 |
Tumor_09/137_HGU133 Plus 2.0+3' IVT kit |
| GSM1904891 |
Tumor_10/12_HGU133 Plus 2.0+3' IVT kit |
| GSM1904892 |
Tumor_08/40_X3P Array+3' IVT kit |
| GSM1904893 |
Tumor_08/95_X3P Array+3' IVT kit |
| GSM1904894 |
Tumor_09/137_X3P Array+3' IVT kit |
| GSM1904895 |
Tumor_10/12_X3P Array+3' IVT kit |
| GSM1904896 |
Tumor_08/40_HGU133 Plus 2.0+Nugen Ovation FFPE WTA System Kit |
| GSM1904897 |
Tumor_08/95_HGU133 Plus 2.0+Nugen Ovation FFPE WTA System Kit |
| GSM1904898 |
Tumor_09/137_HGU133 Plus 2.0+Nugen Ovation FFPE WTA System Kit |
| GSM1904899 |
Tumor_10/12_HGU133 Plus 2.0+Nugen Ovation FFPE WTA System Kit |
| GSM1904900 |
Tumor_08/40_X3P Array+Nugen Ovation FFPE WTA System Kit |
| GSM1904901 |
Tumor_08/95_X3P Array+Nugen Ovation FFPE WTA System Kit |
| GSM1904902 |
Tumor_09/137_X3P Array+Nugen Ovation FFPE WTA System Kit |
| GSM1904903 |
Tumor_10/12_X3P Array+Nugen Ovation FFPE WTA System Kit |
| GSM1904904 |
Matched control of TT01_X3P Array+Nugen Ovation FFPE WTA System Kit |
| GSM1904905 |
Matched control of TT08_X3P Array+Nugen Ovation FFPE WTA System Kit |
| GSM1904906 |
Matched control of TT31_X3P Array+Nugen Ovation FFPE WTA System Kit |
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| Relations |
| BioProject |
PRJNA298333 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE73883_RAW.tar |
83.2 Mb |
(http)(custom) |
TAR (of CEL, CHP) |
| Processed data included within Sample table |
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