RNA Amplification Protocol Leads to Biased Polymerase Chain Reaction Results Especially for Low-Copy Transcripts of Human Bone Marrow-Derived Stromal Cells

RNA Amplification Protocol Leads to Biased Polymerase Chain Reaction Results Especially for Low-Copy Transcripts of Human Bone Marrow-Derived Stromal Cells

The amplification of RNA is becoming increasingly important, as often only limited amounts of cells are available for gene expression analysis. In this study, the gene expression profile of the 39 human homeobox (HOX) genes was analyzed in bone marrow-derived multipotent stromal cells (BM-MSCs) by r...

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Main Authors: Coenen, Carolin, Liedtke, Stefanie, Kogler, Gesine
Format: Online
Language:English
Published: Public Library of Science 2015
Online Access:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4613830/
id pubmed-4613830
recordtype oai_dc
spelling pubmed-46138302015-10-29 RNA Amplification Protocol Leads to Biased Polymerase Chain Reaction Results Especially for Low-Copy Transcripts of Human Bone Marrow-Derived Stromal Cells Coenen, Carolin Liedtke, Stefanie Kogler, Gesine Research Article The amplification of RNA is becoming increasingly important, as often only limited amounts of cells are available for gene expression analysis. In this study, the gene expression profile of the 39 human homeobox (HOX) genes was analyzed in bone marrow-derived multipotent stromal cells (BM-MSCs) by reverse transcription (RT-) and quantitative polymerase chain reaction (qPCR). For further unlimited gene expression analysis, Whole Transcriptome Amplification (WTA) was used to amplify RNA from human BM-MSCs. However, WTA led to biased RT- and qPCR results, and even non-detectability of HOX transcripts compared to non-amplified BM-MSC samples which instead revealed transcription. It is important to note that the same RNA of the respective human BM-MSC line was used for normal cDNA synthesis by standard reverse transcription (non-amplified RT samples) and for cDNA synthesis by WTA (amplified WTA samples). On this account, the different RT- and qPCR results were unexpected applying WTA. The significantly reduced detection of HOX transcripts after WTA has been demonstrated for numerous BM-MSC lines (n = 26) by RT-PCR analysis. Furthermore, undetectable HOX transcripts meaning HOX transcripts of human BM-MSCs that were detected after normal cDNA synthesis, but were no longer detectable after WTA, were consistently observed by qPCR analysis. Finally, qPCR experiments revealed a possible explanation for the differences between amplified and non-amplified BM-MSC samples: an inverse correlation between the biased qPCR results and the low expression level of the respective HOX gene. The PCR analysis of high-copy transcripts like GAPDH or RPL13A revealed unchanged qPCR results after WTA compared to corresponding non-amplified BM-MSC samples. In contrast, WTA led to biased qPCR results for medium-copy HOX transcripts, and even non-detectability of low-copy HOX transcripts of human BM-MSCs resulting in false negative RT- and qPCR data applying WTA. Public Library of Science 2015-10-20 /pmc/articles/PMC4613830/ /pubmed/26485654 http://dx.doi.org/10.1371/journal.pone.0141070 Text en © 2015 Coenen et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
repository_type Open Access Journal
institution_category Foreign Institution
institution US National Center for Biotechnology Information
building NCBI PubMed
collection Online Access
language English
format Online
author Coenen, Carolin
Liedtke, Stefanie
Kogler, Gesine
spellingShingle Coenen, Carolin
Liedtke, Stefanie
Kogler, Gesine
RNA Amplification Protocol Leads to Biased Polymerase Chain Reaction Results Especially for Low-Copy Transcripts of Human Bone Marrow-Derived Stromal Cells
author_facet Coenen, Carolin
Liedtke, Stefanie
Kogler, Gesine
author_sort Coenen, Carolin
title RNA Amplification Protocol Leads to Biased Polymerase Chain Reaction Results Especially for Low-Copy Transcripts of Human Bone Marrow-Derived Stromal Cells
title_short RNA Amplification Protocol Leads to Biased Polymerase Chain Reaction Results Especially for Low-Copy Transcripts of Human Bone Marrow-Derived Stromal Cells
title_full RNA Amplification Protocol Leads to Biased Polymerase Chain Reaction Results Especially for Low-Copy Transcripts of Human Bone Marrow-Derived Stromal Cells
title_fullStr RNA Amplification Protocol Leads to Biased Polymerase Chain Reaction Results Especially for Low-Copy Transcripts of Human Bone Marrow-Derived Stromal Cells
title_full_unstemmed RNA Amplification Protocol Leads to Biased Polymerase Chain Reaction Results Especially for Low-Copy Transcripts of Human Bone Marrow-Derived Stromal Cells
title_sort rna amplification protocol leads to biased polymerase chain reaction results especially for low-copy transcripts of human bone marrow-derived stromal cells
description The amplification of RNA is becoming increasingly important, as often only limited amounts of cells are available for gene expression analysis. In this study, the gene expression profile of the 39 human homeobox (HOX) genes was analyzed in bone marrow-derived multipotent stromal cells (BM-MSCs) by reverse transcription (RT-) and quantitative polymerase chain reaction (qPCR). For further unlimited gene expression analysis, Whole Transcriptome Amplification (WTA) was used to amplify RNA from human BM-MSCs. However, WTA led to biased RT- and qPCR results, and even non-detectability of HOX transcripts compared to non-amplified BM-MSC samples which instead revealed transcription. It is important to note that the same RNA of the respective human BM-MSC line was used for normal cDNA synthesis by standard reverse transcription (non-amplified RT samples) and for cDNA synthesis by WTA (amplified WTA samples). On this account, the different RT- and qPCR results were unexpected applying WTA. The significantly reduced detection of HOX transcripts after WTA has been demonstrated for numerous BM-MSC lines (n = 26) by RT-PCR analysis. Furthermore, undetectable HOX transcripts meaning HOX transcripts of human BM-MSCs that were detected after normal cDNA synthesis, but were no longer detectable after WTA, were consistently observed by qPCR analysis. Finally, qPCR experiments revealed a possible explanation for the differences between amplified and non-amplified BM-MSC samples: an inverse correlation between the biased qPCR results and the low expression level of the respective HOX gene. The PCR analysis of high-copy transcripts like GAPDH or RPL13A revealed unchanged qPCR results after WTA compared to corresponding non-amplified BM-MSC samples. In contrast, WTA led to biased qPCR results for medium-copy HOX transcripts, and even non-detectability of low-copy HOX transcripts of human BM-MSCs resulting in false negative RT- and qPCR data applying WTA.
publisher Public Library of Science
publishDate 2015
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4613830/
_version_ 1613490741460336640

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