Evaluation of putative internal reference genes for gene expression normalization in Nannochloropsis sp. by quantitative real-time RT-PCR

2012 ◽  
Vol 424 (1) ◽  
pp. 118-123 ◽  
Author(s):  
Shaona Cao ◽  
Xiaowen Zhang ◽  
Naihao Ye ◽  
Xiao Fan ◽  
Shanli Mou ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Reference Genes ◽  
Rt Pcr ◽  
Internal Reference

scholarly journals Gene Expression Studies: How to Obtain Accurate and Reliable Data by Quantitative Real-Time RT PCR / IZUČAVANJE EKSPRESIJE GENA: KAKO DOBITI TAČNE I POUZDANE PODATKE KVANTITATIVNIM RT PCR-OM U REALNOM VREMENU

2013 ◽  
Vol 32 (4) ◽  
pp. 325-338 ◽  
Author(s):  
Jelena Nestorov ◽  
Gordana Matić ◽  
Ivana Elaković ◽  
Nikola Tanić
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Reference Genes ◽  
Dynamic Range ◽  
Rna Extraction ◽  
Housekeeping Genes ◽  
Reliable Data ◽  
Endogenous Control ◽  
Rt Pcr ◽  
Internal Reference

Summary Real-time RT PCR has been recognized as an accurate, reliable and sensitive method for quantifying gene transcription. However, several steps preceding PCR represent critical points and source of inaccuracies. These steps include cell processing, RNA extraction, RNA storage, assessment of RNA concentration and cDNA synthesis. To compensate for potential variability introduced by the procedure, normalization of target gene expression has been established. Accurate normalization has become an absolute prerequisite for the correct quantification of gene expression. Several strategies are in use for the normalization of data, including normalization to sample size, to total RNA or to an internal reference. Among these, the use of housekeeping genes as an internal (endogenous) control is the most common approach. Given the increased sensitivity, reproducibility and large dynamic range of this methodology, the requirements for a proper reference gene for normalization have become increasingly stringent. The aim of this paper is to discuss the concept of normalization in mRNA quantification, as well as to discuss several statistical algorithms developed to help the validation of potential reference genes. By showing that the use of inappropriate endogenous control might lead to incorrect results and misinterpretation of experimental data, we are joining the creators of Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) in an attempt to convince scientists that proper validation of potential reference genes is an absolute prerequisite for correct normalization and, therefore, for providing accurate and reliable data by quantitative real-time RT PCR gene expression analyses.

Per-unit-living tissue normalization of real-time RT-PCR data in ischemic rat hearts

2012 ◽  
Vol 44 (12) ◽  
pp. 651-656 ◽  
Author(s):  
S. Ellefsen ◽  
M. Bliksøen ◽  
A. Rutkovskiy ◽  
I. B. Johansen ◽  
M.-L. Kaljusto ◽  
...  
Keyword(s):  
Infarct Size ◽  
Real Time ◽  
Reference Genes ◽  
Stable Expression ◽  
Unit Weight ◽  
Living Tissue ◽  
Rt Pcr ◽  
Internal Reference ◽  
Total Rna ◽  
Rat Hearts

In studies of gene expression in acute ischemic heart tissue, internal reference genes need to show stable expression per-unit-living tissue to hinder dead cells from biasing real-time RT-PCR data. Until now, this important issue has not been appropriately investigated. We hypothesized that the expression of seven internal reference genes would show stable per-unit-living tissue expression in Langendorff-perfused rat hearts subjected to ischemia-reperfusion. This was found for cyclophilin A, GAPDH, RPL-32, and PolR2A mRNA, with GAPDH showing the highest degree of stability ( R = 0.11), suggesting unchanged rates of mRNA transcription in live cells and complete degradation of mRNA from dead cells. The infarct size-dependent degradation of GAPDH was further supported by a close correlation between changes in GAPDH mRNA and changes in RNA quality measured as RNA integrity number (R = 0.90, P < 0.05). In contrast, β-actin and 18S rRNA showed stable expression per-unit-weight tissue and a positive correlation with infarct size (R = 0.61 and R = 0.77, P < 0.05 for both analyses). The amount of total RNA extracted per-unit-weight tissue did not differ between groups despite wide variation in infarct size (7.1–50.1%). When β-actin expression was assessed using four different normalization strategies, GAPDH and geNorm provided appropriate per-unit-living expression, while 18S and total RNA resulted in marked underestimations. In studies of ischemic tissues, we recommend using geometric averaging of carefully selected reference genes for normalization of real-time RT-PCR data. A marked shift in the mRNA/rRNA ratio renders rRNA as useless for normalization purposes.

Reference genes for gene expression normalization in Chlamydomonas sp. ICE-L by quantitative real-time RT-PCR

2014 ◽  
Vol 24 (3) ◽  
pp. 276-282 ◽  
Author(s):  
Shanli Mou ◽  
Xiaowen Zhang ◽  
Jinlai Miao ◽  
Zhou Zheng ◽  
Dong Xu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Reference Genes ◽  
Rt Pcr ◽  
Chlamydomonas Sp

scholarly journals Validation of reference genes for gene expression studies in the honey bee,Apis mellifera, by quantitative real-time RT-PCR

Apidologie ◽  
2008 ◽  
Vol 39 (3) ◽  
pp. 372-385 ◽  
Author(s):  
Anete Pedro Lourenço ◽  
Aline Mackert ◽  
Alexandre dos Santos Cristino ◽  
Zilá Luz Paulino Simões
Keyword(s):  
Gene Expression ◽  
Apis Mellifera ◽  
Real Time ◽  
Honey Bee ◽  
Reference Genes ◽  
Rt Pcr ◽  
Expression Studies ◽  
Gene Expression Studies

scholarly journals Validation of Reference Genes for Gene Expression by Quantitative Real-Time RT-PCR in Stem Segments Spanning Primary to Secondary Growth in Populus tomentosa

PLoS ONE ◽  
2016 ◽  
Vol 11 (6) ◽  
pp. e0157370 ◽  
Author(s):  
Ying Wang ◽  
Yajuan Chen ◽  
Liping Ding ◽  
Jiewei Zhang ◽  
Jianhua Wei ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Reference Genes ◽  
Secondary Growth ◽  
Populus Tomentosa ◽  
Rt Pcr ◽  
Stem Segments

scholarly journals Standardization of Gene Expression Quantification by Absolute Real-Time qRT-PCR System Using a Single Standard for Marker and Reference Genes

2010 ◽  
Vol 5 ◽  
pp. BMI.S5596 ◽  
Author(s):  
Yi-Hong Zhou ◽  
Vinay R. Raj ◽  
Eric Siegel ◽  
Liping Yu
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Gene Expression Data ◽  
Reference Genes ◽  
Pcr Analysis ◽  
Expression Data ◽  
Internal Reference ◽  
Expression Ratio ◽  
Qrt Pcr ◽  
Single Standard

In the last decade, genome-wide gene expression data has been collected from a large number of cancer specimens. In many studies utilizing either microarray-based or knowledge-based gene expression profiling, both the validation of candidate genes and the identification and inclusion of biomarkers in prognosis-modeling has employed real-time quantitative PCR on reverse transcribed mRNA (qRT-PCR) because of its inherent sensitivity and quantitative nature. In qRT-PCR data analysis, an internal reference gene is used to normalize the variation in input sample quantity. The relative quantification method used in current real-time qRT-PCR analysis fails to ensure data comparability pivotal in identification of prognostic biomarkers. By employing an absolute qRT-PCR system that uses a single standard for marker and reference genes (SSMR) to achieve absolute quantification, we showed that the normalized gene expression data is comparable and independent of variations in the quantities of sample as well as the standard used for generating standard curves. We compared two sets of normalized gene expression data with same histological diagnosis of brain tumor from two labs using relative and absolute real-time qRT-PCR. Base-10 logarithms of the gene expression ratio relative to ACTB were evaluated for statistical equivalence between tumors processed by two different labs. The results showed an approximate comparability for normalized gene expression quantified using a SSMR-based qRT-PCR. Incomparable results were seen for the gene expression data using relative real-time qRT-PCR, due to inequality in molar concentration of two standards for marker and reference genes. Overall results show that SSMR-based real-time qRT-PCR ensures comparability of gene expression data much needed in establishment of prognostic/predictive models for cancer patients–-a process that requires large sample sizes by combining independent sets of data.

Evaluation of reference genes for quantitative real-time RT-PCR analysis of gene expression in Nile tilapia (Oreochromis niloticus)

Gene ◽  
2013 ◽  
Vol 527 (1) ◽  
pp. 183-192 ◽  
Author(s):  
Chang Geng Yang ◽  
Xian Li Wang ◽  
Juan Tian ◽  
Wei Liu ◽  
Fan Wu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Oreochromis Niloticus ◽  
Reference Genes ◽  
Nile Tilapia ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Nile Tilapia Oreochromis Niloticus
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