Strategy of the use of 28S rRNA as a housekeeping gene in real-time quantitative PCR analysis of gene transcription in insect cells infected by viruses

2010 ◽  
Vol 163 (2) ◽  
pp. 210-215 ◽  
Author(s):  
Jian-Li Xue ◽  
Tamer Z. Salem ◽  
Colin M. Turney ◽  
Xiao-Wen Cheng
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Gene Transcription ◽  
Insect Cells ◽  
Housekeeping Gene ◽  
Pcr Analysis ◽  
28S Rrna ◽  
Real Time Quantitative Pcr

Real-time RT-PCR analysis of housekeeping genes in human skeletal muscle following acute exercise

2004 ◽  
Vol 18 (2) ◽  
pp. 226-231 ◽  
Author(s):  
Douglas J. Mahoney ◽  
Kate Carey ◽  
Ming-Hua Fu ◽  
Rodney Snow ◽  
David Cameron-Smith ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Real Time ◽  
Healthy Subjects ◽  
Acute Exercise ◽  
Housekeeping Genes ◽  
Housekeeping Gene ◽  
Cycle Ergometry ◽  
Pcr Analysis ◽  
28S Rrna ◽  
Rt Pcr

Studies examining gene expression with RT-PCR typically normalize their mRNA data to a constitutively expressed housekeeping gene. The validity of a particular housekeeping gene must be determined for each experimental intervention. We examined the expression of various housekeeping genes following an acute bout of endurance (END) or resistance (RES) exercise. Twenty-four healthy subjects performed either a interval-type cycle ergometry workout to exhaustion (∼75 min; END) or 300 single-leg eccentric contractions (RES). Muscle biopsies were taken before exercise and 3 h and 48 h following exercise. Real-time RT-PCR was performed on β-actin, cyclophilin (CYC), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β2-microglobulin (β2M). In a second study, 10 healthy subjects performed 90 min of cycle ergometry at ∼65% of V̇o2 max, and we examined a fifth housekeeping gene, 28S rRNA, and reexamined β2M, from muscle biopsy samples taken immediately postexercise. We showed that CYC increased 48 h following both END and RES exercise (3- and 5-fold, respectively; P < 0.01), and 28S rRNA increased immediately following END exercise (2-fold; P = 0.02). β-Actin trended toward an increase following END exercise (1.85-fold collapsed across time; P = 0.13), and GAPDH trended toward a small yet robust increase at 3 h following RES exercise (1.4-fold; P = 0.067). In contrast, β2M was not altered at any time point postexercise. We conclude that β2M and β-actin are the most stably expressed housekeeping genes in skeletal muscle following RES exercise, whereas β2M and GAPDH are the most stably expressed following END exercise.

Selection of Stable Reference Genes for Real-Time Quantitative PCR Analysis in Edwardsiella tarda

2017 ◽  
Vol 27 (1) ◽  
pp. 112-121 ◽  
Author(s):  
Zhongyang Sun ◽  
Jia Deng ◽  
Haizhen Wu ◽  
Qiyao Wang ◽  
Yuanxing Zhang
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Reference Genes ◽  
Edwardsiella Tarda ◽  
Pcr Analysis ◽  
Real Time Quantitative Pcr ◽  
Selection Of

Human disease characterization: real-time quantitative PCR analysis of gene expression

2001 ◽  
Vol 6 (20) ◽  
pp. 1062-1067 ◽  
Author(s):  
James V Snider ◽  
Mark A Wechser ◽  
Izidore S Lossos
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Quantitative Pcr ◽  
Human Disease ◽  
Pcr Analysis ◽  
Real Time Quantitative Pcr

scholarly journals Establishing reference genes for use in real-time quantitative PCR analysis of early equine embryos

2011 ◽  
Vol 23 (2) ◽  
pp. 353 ◽  
Author(s):  
Damien B. B. P. Paris ◽  
Ewart W. Kuijk ◽  
Bernard A. J. Roelen ◽  
Tom A. E. Stout
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Quantitative Pcr ◽  
Embryo Development ◽  
Reference Genes ◽  
Stable Expression ◽  
Pcr Analysis ◽  
Blastocyst Development ◽  
Real Time Quantitative Pcr

Real-time quantitative PCR (qPCR) is invaluable for investigating changes in gene expression during early development, since it can be performed on the limited quantities of mRNA contained in individual embryos. However, the reliability of this method depends on the use of validated stably expressed reference genes for accurate data normalisation. The aim of the present study was to identify and validate a set of reference genes suitable for studying gene expression during equine embryo development. The stable expression of four carefully selected reference genes and one developmentally regulated gene was examined by qPCR in equine in vivo embryos from morula to expanded blastocyst stage. SRP14, RPL4 and PGK1 were identified by geNorm analysis as stably expressed reference genes suitable for data normalisation. RPL13A expression was less stable and changed significantly during the period of development examined, rendering it unsuitable as a reference gene. As anticipated, CDX2 expression increased significantly during embryo development, supporting its possible role in trophectoderm specification in the horse. In summary, it was demonstrated that evidence-based selection of potential reference genes can reduce the number needed to validate stable expression in an experimental system; this is particularly useful when dealing with tissues that yield small amounts of mRNA. SRP14, RPL4 and PGK1 are stable reference genes suitable for normalising expression for genes of interest during in vivo morula to expanded blastocyst development of horse embryos.

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