{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":["Santra Santhosh"],"organism":["Homo sapiens"],"software":["NimbleScan 2.1"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-15163"],"description":["Gene expression microarrays require synthesizing a vast number of unique 60mer high-fidelity DNA probes to achieve strong signal intensity and low background noise, which are essential for accurate data interpretation. Due to the high number of coupling cycles involved in the synthesis, these DNA probes are susceptible to degradation, especially if synthesized in the absence of a debranching/capping step. This study compared gene expression microarrays synthesized with and without incorporating the debranching step to evaluate their impact on probe quality and the detection sensitivity for the genes expressed above the background levels."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Labeling - The mRNAs from the total human reference RNA pool was reverse transcribed to cDNA and Cy3-labelled by following Ouelett et al's protocol, which is described as the following.  A 18.5 μL solution containing 10 μg of total RNA, 7 μg of 5′-Cy3-labeled random nonamers (Biomers), and 1.5 μL of nuclease-free water was incubated at 70 °C for 5 min for heat denaturation and the solution was immediately cooled on ice. 6 μL of 5× first-strand buffer (Invitrogen), 1.5 μL of 0.1 M DTT (Invitrogen), 1 μL of 10 mM dNTP (Thermo Scientific), 4 μL of 200 U/μL superscript III (invitrogen), and 1 μL of 40 U/μL RNaseOUT (Invitrogen) was mixed to prepare 13.5 μL of reverse-transcription buffer. To this, the denatured solution was added and incubated at 25 °C for 5 min, followed by a 3 h incubation at 42 °C. This was followed by adding a hydrolysis reaction mixture containing 32 μL solution of 200 mM NaOH and 200 mM EDTA in 1:1 ratio. This was then incubated at 65 °C for 10 min. 64 μL of 1 M HEPES at pH 7 (Carl Roth) was added to the mixture to neutralize the reaction solution. QiaQuick PCR purification kit  (Qiagen) was used to purify the sample following the manufacturer's protocol. The amount of Cy3 labelled cDNA was measured using a NanoDrop One (ThermoFisher Scientific).","Nucleic Acid Extraction - This gene expression microarray experiment is designed to evaluate the quality of DNA probes synthesized directly on the microarray surface with different synthesis methods, rather than to assess differential gene expression of different biological conditions. therefore, it does not require cDNA samples from different treatments or experimental groups. Here For the same reason, we do not require cDNA samples with different treatment. To maintain consistency and eliminate variables related to biological conditions we have used Cy3-labelled cDNA prepared by reverse transcription of Universal Human Reference RNA (Agilent, 74000) and used uniformly across all arrays.","Scaning - All the arrays were scanned with Innopsys Innoscan 1100 scanner at 532 nm and 2 µm resolution. The fluorescence signal intensities were extracted using NimbleScan 2.1 software.","Sample Collection - This gene expression microarray experiment is designed to evaluate the quality of DNA probes synthesized directly on the microarray surface with different synthesis methods, rather than to assess differential gene expression of different biological conditions. therefore, it does not require cDNA samples from different treatments or experimental groups. Here For the same reason, we do not require cDNA samples with different treatment. To maintain consistency and eliminate variables related to biological conditions we have used Cy3-labelled cDNA prepared by reverse transcription of Universal Human Reference RNA (Agilent, 74000) and used uniformly across all arrays.","Hybridization - 300 µl of hybridization mixture containing 5 µg of Cy3-labelled cDNA in 150 µl 2x MES buffer, 8.34 µl of 20 mg/ml of acetylated BSA (Invitrogen AM2614), 3.33 µl of 10 mg/ml herring-sperm DNA, 11.11 µl each of 100 nM of Cy3-labelled synthetic spike-in controls \\\"EcoBioD2_60mer\\\", \\\"EcoBioA1t_53mer\\\" and QC25mer\" was hybridized on the gene expression microarrays in a SecureSeal hybridization chamber (Grace Bio-Labs SA200) for 21 hours at 42 °C.  The hybridized arrays were washed with Non-Stringent Wash Buffer (NSWB; 6× SSPE, 0.1% Tween20) for 2 minute, Stringent Wash Buffer (SWB; 100 mM MES, 0.1 M Na+, 0.01% Tween20) for 1 minute and Final Wash Buffer (FWB; 0.1× SSC) for a few seconds and dried with microarray centrifuge."],"figure_sub":["MIAME Score","Raw Data","Organization","Assays and Data","Processed Data","MAGE-TAB Files","Array Designs"],"data_protocol":["Data Transformation - To compare the array performance, the expression data of all the 4 arrays were normalized together using Robust Multichip Analysis (RMA) provided by NimbleScan 2.1.  To compare the overall intensity improvement by incorporating debranching step in the synthesis, the set of technical replicates were normalized using RMA and compared."],"omics_type":["Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["Innopsys Innoscan"],"study_type":["transcription profiling by array"],"species":["Homo sapiens"],"pubmed_title":["Digital Photolithographic Synthesis of Large, High-Quality DNA Libraries and Microarrays via an Efficient Debranching Strategy"],"pubmed_authors":["Santra Santhosh, Sharon Istvánffy, Maya Giridhar, Jürgen Behr, Mark M. Somoza","Santra Santhosh","Mark Somoza"],"additional_accession":[]},"is_claimable":false,"name":"Comparison of Microarray Synthesis with and without Incorporating Debranching/Capping Step via Gene Expression Analysis","description":"Gene expression microarrays require synthesizing a vast number of unique 60mer high-fidelity DNA probes to achieve strong signal intensity and low background noise, which are essential for accurate data interpretation. Due to the high number of coupling cycles involved in the synthesis, these DNA probes are susceptible to degradation, especially if synthesized in the absence of a debranching/capping step. This study compared gene expression microarrays synthesized with and without incorporating the debranching step to evaluate their impact on probe quality and the detection sensitivity for the genes expressed above the background levels.","dates":{"release":"2025-06-30T00:00:00Z","modification":"2025-05-27T08:38:14.753Z","creation":"2025-05-27T08:38:14.753Z"},"accession":"E-MTAB-15163","cross_references":{"EFO":["EFO_0002768","EFO_0003814","EFO_0002944","EFO_0003813","EFO_0003816","EFO_0005518","EFO_0003815"]}}