MicroRNAs and Reference Gene Methodology

2019 ◽  
pp. 2233-2250
Author(s):  
Petra Matoušková
Keyword(s):  
Reference Gene

scholarly journals Optimizations for identifying reference genes in bone and cartilage bioengineering

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Fei Xiong ◽  
Xiangyun Cheng ◽  
Chao Zhang ◽  
Roland Manfred Klar ◽  
Tao He
Keyword(s):  
Gene Expression ◽  
Skeletal Muscle ◽  
Adipose Tissue ◽  
Real Time ◽  
Reference Gene ◽  
Muscle Tissue ◽  
Reference Genes ◽  
Target Genes ◽  
The Stability ◽  
And Cartilage

Abstract Background Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) remains one of the best-established techniques to assess gene expression patterns. However, appropriate reference gene(s) selection remains a critical and challenging subject in which inappropriate reference gene selction can distort results leading to false interpretations. To date, mixed opinions still exist in how to choose the most optimal reference gene sets in accodrance to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guideline. Therefore, the purpose of this study was to investigate which schemes were the most feasible for the identification of reference genes in a bone and cartilage bioengineering experimental setting. In this study, rat bone mesenchymal stem cells (rBMSCs), skeletal muscle tissue and adipose tissue were utilized, undergoing either chondrogenic or osteogenic induction, to investigate the optimal reference gene set identification scheme that would subsequently ensure stable and accurate interpretation of gene expression in bone and cartilage bioengineering. Results The stability and pairwise variance of eight candidate reference genes were analyzed using geNorm. The V0.15- vs. Vmin-based normalization scheme in rBMSCs had no significant effect on the eventual normalization of target genes. In terms of the muscle tissue, the results of the correlation of NF values between the V0.15 and Vmin schemes and the variance of target genes expression levels generated by these two schemes showed that different schemes do indeed have a significant effect on the eventual normalization of target genes. Three selection schemes were adopted in terms of the adipose tissue, including the three optimal reference genes (Opt3), V0.20 and Vmin schemes, and the analysis of NF values with eventual normalization of target genes showed that the different selection schemes also have a significant effect on the eventual normalization of target genes. Conclusions Based on these results, the proposed cut-off value of Vn/n + 1 under 0.15, according to the geNorm algorithm, should be considered with caution. For cell only experiments, at least rBMSCs, a Vn/n + 1 under 0.15 is sufficient in RT-qPCR studies. However, when using certain tissue types such as skeletal muscle and adipose tissue the minimum Vn/n + 1 should be used instead as this provides a far superior mode of generating accurate gene expression results. We thus recommended that when the stability and variation of a candidate reference genes in a specific study is unclear the minimum Vn/n + 1 should always be used as this ensures the best and most accurate gene expression value is achieved during RT-qPCR assays.

scholarly journals Cloning Reference Gene rpoB in Serratia plymuthica UBCF_13

2021 ◽  
Vol 741 (1) ◽  
pp. 012040
Author(s):  
R Trivano ◽  
R Fatiah ◽  
J Jamsari
Keyword(s):  
Reference Gene ◽  
Serratia Plymuthica

scholarly journals A Multiplex, Droplet Digital PCR Assay for the Detection of T-Cell Receptor Excision Circles and Kappa-Deleting Recombination Excision Circles

2019 ◽  
Vol 66 (1) ◽  
pp. 229-238 ◽  
Author(s):  
Tracie Profaizer ◽  
Patricia Slev
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
T Cell Receptor ◽  
Reference Gene ◽  
Cell Receptor ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Blood Samples ◽  
Healthy Donors ◽  
Excision Circles

Abstract BACKGROUND T-cell receptor excision circles (TREC) and κ-deleting recombination receptor excision circles (KREC) concentrations can be used to assess and diagnose immune deficiencies, monitor thymic and bone marrow immune reconstitution, or follow responses to drug therapy. We developed an assay to quantify TREC, KREC, and a reference gene in a single reaction using droplet digital PCR (ddPCR). METHODS PCR was optimized for 3 targets: TREC, KREC, and ribonuclease P/MRP subunit p30 (RPP30) as the reference gene. Multiplexing was accomplished by varying the target's fluorophore and concentration. Correlation with clinical results was evaluated using 47 samples from healthy donors, 59 samples with T-cell and B-cell markers within the reference interval from the flow cytometry laboratory, 20 cord blood samples, and 34 samples submitted for exome sequencing for severe combined immunodeficiency disease (SCID). RESULTS The limit of the blank was 4 positive droplets, limit of detection 9 positive droplets, and limit of quantification 25 positive droplets, or 2.0 copies/μL. TREC and KREC copies/μL were as expected in the healthy donors and cord blood samples and concordant with the healthy flow cytometry results. Of the samples from the SCID Panel, 56.5% had a TREC count <20 copies/μL and 17.7% had a KREC count <20 copies/μL, suggestive of low T- and B-cell numbers, respectively. CONCLUSIONS Our multiplex ddPCR assay is an analytically sensitive and specific method for the absolute quantification of TREC and KREC. To the best of our knowledge, this paper is the first to describe the simultaneous quantification of TREC, KREC, and a reference gene by use of ddPCR.

scholarly journals Current Practices for Reference Gene Selection in RT-qPCR of Aspergillus: Outlook and Recommendations for the Future

Genes ◽  
2021 ◽  
Vol 12 (7) ◽  
pp. 960
Author(s):  
Meagan Archer ◽  
Jianping Xu
Keyword(s):  
Gene Expression ◽  
Reference Gene ◽  
Reference Genes ◽  
Gene Selection ◽  
Quantitative Polymerase Chain Reaction ◽  
Specific Method ◽  
Experimental Conditions ◽  
Reference Gene Selection ◽  
Physiological Processes ◽  
The Stability

Aspergillus is a genus of filamentous fungi with vast geographic and ecological distributions. Species within this genus are clinically, agriculturally and biotechnologically relevant, leading to increasing interest in elucidating gene expression dynamics of key metabolic and physiological processes. Reverse-transcription quantitative Polymerase Chain Reaction (RT-qPCR) is a sensitive and specific method of quantifying gene expression. A crucial step for comparing RT-qPCR results between strains and experimental conditions is normalisation to experimentally validated reference gene(s). In this review, we provide a critical analysis of current reference gene selection and validation practices for RT-qPCR gene expression analyses of Aspergillus. Of 90 primary research articles obtained through our PubMed query, 17 experimentally validated the reference gene(s) used. Twenty reference genes were used across the 90 studies, with beta-tubulin being the most used reference gene, followed by actin, 18S rRNA and glyceraldehyde 3-phosphate dehydrogenase. Sixteen of the 90 studies used multiple reference genes for normalisation. Failing to experimentally validate the stability of reference genes can lead to conflicting results, as was the case for four studies. Overall, our review highlights the need to experimentally validate reference genes in RT-qPCR studies of Aspergillus.

scholarly journals Identification and selection of reference genes for gene expression analysis by quantitative real-time PCR in Suaeda glauca’s response to salinity

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Meng Wang ◽  
Tingting Ren ◽  
Prince Marowa ◽  
Haina Du ◽  
Zongchang Xu
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Reference Gene ◽  
Reference Genes ◽  
Suitable Reference Gene ◽  
Normalize Gene Expression ◽  
Suaeda Glauca ◽  
Suitable Reference ◽  
Germinating Seeds ◽  
Different Tissues

AbstractQuantitative real-time polymerase chain reaction (qPCR) using a stable reference gene is widely used for gene expression research. Suaeda glauca L. is a succulent halophyte and medicinal plant that is extensively used for phytoremediation and extraction of medicinal compounds. It thrives under high-salt conditions, which promote the accumulation of high-value secondary metabolites. However, a suitable reference gene has not been identified for gene expression standardization in S. glauca under saline conditions. Here, 10 candidate reference genes, ACT7, ACT11, CCD1, TUA5, UPL1, PP2A, DREB1D, V-H+-ATPase, MPK6, and PHT4;5, were selected from S. glauca transcriptome data. Five statistical algorithms (ΔCq, geNorm, NormFinder, BestKeeper, and RefFinder) were applied to determine the expression stabilities of these genes in 72 samples at different salt concentrations in different tissues. PP2A and TUA5 were the most stable reference genes in different tissues and salt treatments, whereas DREB1D was the least stable. The two reference genes were sufficient to normalize gene expression across all sample sets. The suitability of identified reference genes was validated with MYB and AP2 in germinating seeds of S. glauca exposed to different NaCl concentrations. Our study provides a foundational framework for standardizing qPCR analyses, enabling accurate gene expression profiling in S. glauca.

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