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使用定量实时聚合酶链反应验证用于胶质母细胞瘤基因表达分析的管家基因

Validation of housekeeping genes for gene expression analysis in glioblastoma using quantitative real-time polymerase chain reaction.

作者信息

Aithal Madhuri G S, Rajeswari Narayanappa

机构信息

Department of Biotechnology, Dayananda Sagar College of Engineering, Bangalore, India.

出版信息

Brain Tumor Res Treat. 2015 Apr;3(1):24-9. doi: 10.14791/btrt.2015.3.1.24. Epub 2015 Apr 29.

DOI:10.14791/btrt.2015.3.1.24
PMID:25977903
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC4426273/
Abstract

BACKGROUND

Quantitative real-time polymerase chain reaction (qPCR) is the most reliable tool for gene expression studies. Selection of housekeeping genes (HKGs) that are having most stable expression is critical to carry out accurate gene expression profiling. There is no 'universal' HKG having stable expression in all kinds of tissues under all experimental conditions.

METHODS

The present study aims to identify most appropriate HKGs for gene expression analysis in glioblastoma (GBM) samples. Based on literature survey, six most commonly used HKGs that are invariant in GBM were chosen. We performed qPCR using RNA from formalin fixed paraffin embedded GBM samples and normal brain samples to investigate the expression pattern of HPRT, GAPDH, TBP, B2M, B2M, RPL13A, and RN18S1 with different abundance. A simple Δcycle threshold approach was employed to calculate the fold change.

RESULTS

Our study shows that the expression of RPL13A and TBP were found to be most stable across all the samples and are thus suitable for gene expression analysis in human GBM. Except for TBP, none of the other conventionally used HKGs in GBM studies e.g., HPRT and GAPDH were found to be suitable as they showed variation in RNA expression.

CONCLUSION

Validation of HKGs is therefore immensely specific for a particular experimental setup and is crucial in assessing any new setup.

摘要

背景

定量实时聚合酶链反应（qPCR）是基因表达研究中最可靠的工具。选择具有最稳定表达的管家基因（HKGs）对于进行准确的基因表达谱分析至关重要。不存在在所有实验条件下在各种组织中都具有稳定表达的“通用”HKG。

方法

本研究旨在确定胶质母细胞瘤（GBM）样本中用于基因表达分析的最合适的HKGs。基于文献调查，选择了在GBM中不变的六种最常用的HKGs。我们使用来自福尔马林固定石蜡包埋的GBM样本和正常脑样本的RNA进行qPCR，以研究具有不同丰度的次黄嘌呤磷酸核糖转移酶（HPRT）、甘油醛-3-磷酸脱氢酶（GAPDH）、TATA盒结合蛋白（TBP）、β2微球蛋白（B2M）、核糖体蛋白L13A（RPL13A）和18S核糖体RNA（RN18S1）的表达模式。采用简单的Δ循环阈值方法计算倍数变化。

结果

我们的研究表明，RPL13A和TBP的表达在所有样本中最稳定，因此适用于人类GBM中的基因表达分析。除了TBP外，GBM研究中其他常用的HKGs，如HPRT和GAPDH，均未发现适合，因为它们的RNA表达存在差异。

结论

因此，HKGs的验证对于特定的实验设置极具特异性，并且在评估任何新设置时至关重要。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5d02/4426273/a842c67f4f6f/btrt-3-24-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5d02/4426273/0a9ff7d8c22a/btrt-3-24-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5d02/4426273/a842c67f4f6f/btrt-3-24-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5d02/4426273/0a9ff7d8c22a/btrt-3-24-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5d02/4426273/a842c67f4f6f/btrt-3-24-g002.jpg

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