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fISHing with immunohistochemistry for housekeeping gene changes in Alzheimer’s disease using an automated quantitative analysis workflow

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Title: fISHing with immunohistochemistry for housekeeping gene changes in Alzheimer’s disease using an automated quantitative analysis workflow
Authors: Highet, Blake, Vikas Anekal, Praju, Ryan, Brigid, Murray, Helen, Coppieters't Wallant, Natacha, Victor Dieriks, Birger, Singh-Bains, Malvindar K, Mehrabi, Nasim F, Faull, Richard L M, Dragunow, Michael, Curtis, Maurice A
Contributors: Brain Research New Zealand, Neurological Foundation of New Zealand, Health Research Council of New Zealand
Source: Journal of Neurochemistry. 157:1270-1283
Publisher Information: Wiley, 2021.
Publication Year: 2021
Subject Terms: Male, 0301 basic medicine, Messenger, Cyclophilins/analysis, Alzheimer's Disease, Biochemistry, Workflow, Cyclophilins, Essential, Immunohistochemistry/methods, 80 and over, High-Throughput Screening Assays/methods, Human health sciences, In Situ Hybridization, In Situ Hybridization, Fluorescence, Aged, 80 and over, 0303 health sciences, Genes, Essential, RNA, Messenger/analysis, In Situ Hybridization, Fluorescence/methods, DNA-Directed RNA Polymerases, Middle Aged, Immunohistochemistry, 3. Good health, Neurology, immunohistochemistry, Female, cyclophilin B, housekeeping genes, POLR2A RNA polymerase, human, automated analysis workflow, Sciences de la santé humaine, Fluorescence, Cellular and Molecular Neuroscience, 03 medical and health sciences, ubiquitin C (UBC), Alzheimer Disease, Neurologie, Humans, RNA, Messenger, DNA-Directed RNA Polymerases/analysis, Aged, Gene Expression Profiling, Ubiquitin C/analysis, High-Throughput Screening Assays, Genes, RNA, Ubiquitin C, in situ hybridization, Gene Expression Profiling/methods, Transcriptome
Description: In situ hybridization (ISH) is a powerful tool that can be used to localize mRNA expression in tissue samples. Combining ISH with immunohistochemistry (IHC) to determine cell type provides cellular context of mRNA expression, which cannot be achieved with gene microarray or polymerase chain reaction. To study mRNA and protein expression on the same section we investigated the use of RNAscope® ISH in combination with fluorescent IHC on paraffin‐embedded human brain tissue. We first developed a high‐throughput, automated image analysis workflow for quantifying RNA puncta across the total cell population and within neurons identified by NeuN+ immunoreactivity. We then applied this automated analysis to tissue microarray (TMA) sections of middle temporal gyrus tissue (MTG) from neurologically normal and Alzheimer's Disease (AD) cases to determine the suitability of three commonly used housekeeping genes: ubiquitin C (UBC), peptidyl‐prolyl cis‐trans isomerase B (PPIB) and DNA‐directed RNA polymerase II subunit RPB1 (POLR2A). Overall, we saw a significant decrease in total and neuronal UBC expression in AD cases compared to normal cases. Total expression results were validated with RT‐qPCR using fresh frozen tissue from 5 normal and 5 AD cases. We conclude that this technique combined with our novel automated analysis pipeline provides a suitable platform to study changes in gene expression in diseased human brain tissue with cellular and anatomical context. Furthermore, our results suggest that UBC is not a suitable housekeeping gene in the study of post‐mortem AD brain tissue. image
Document Type: Article
File Description: Print-Electronic; application/pdf
Language: English
ISSN: 1471-4159
0022-3042
DOI: 10.1111/jnc.15283
Access URL: https://pubmed.ncbi.nlm.nih.gov/33368239
https://www.ncbi.nlm.nih.gov/pubmed/33368239
http://www.ncbi.nlm.nih.gov/pubmed/33368239
https://pubmed.ncbi.nlm.nih.gov/33368239/
https://onlinelibrary.wiley.com/doi/10.1111/jnc.15283
Rights: Wiley Online Library User Agreement
Accession Number: edsair.doi.dedup.....b0c56a19e000b424da90b1389a82c7cf
Database: OpenAIRE
Description
Abstract:In situ hybridization (ISH) is a powerful tool that can be used to localize mRNA expression in tissue samples. Combining ISH with immunohistochemistry (IHC) to determine cell type provides cellular context of mRNA expression, which cannot be achieved with gene microarray or polymerase chain reaction. To study mRNA and protein expression on the same section we investigated the use of RNAscope® ISH in combination with fluorescent IHC on paraffin‐embedded human brain tissue. We first developed a high‐throughput, automated image analysis workflow for quantifying RNA puncta across the total cell population and within neurons identified by NeuN+ immunoreactivity. We then applied this automated analysis to tissue microarray (TMA) sections of middle temporal gyrus tissue (MTG) from neurologically normal and Alzheimer's Disease (AD) cases to determine the suitability of three commonly used housekeeping genes: ubiquitin C (UBC), peptidyl‐prolyl cis‐trans isomerase B (PPIB) and DNA‐directed RNA polymerase II subunit RPB1 (POLR2A). Overall, we saw a significant decrease in total and neuronal UBC expression in AD cases compared to normal cases. Total expression results were validated with RT‐qPCR using fresh frozen tissue from 5 normal and 5 AD cases. We conclude that this technique combined with our novel automated analysis pipeline provides a suitable platform to study changes in gene expression in diseased human brain tissue with cellular and anatomical context. Furthermore, our results suggest that UBC is not a suitable housekeeping gene in the study of post‐mortem AD brain tissue. image
ISSN:14714159
00223042
DOI:10.1111/jnc.15283