Optimization of Annealing Temperature of HIF-1 A and 18s rRNA in Blood of Swimming Athletes Using RT-PCR

DOI

10.2991/absr.k.200807.008How to use a DOI?

Keywords

in silico, optimization, HIF-1, 18s rRNA, annealing temperature

Abstract

Gene expression is the process of reading from DNA into a functional product. Regulation of gene expression through complex mechanisms. HIF1-1α is one of the regulatory genes that regulates the return of homeostasis when there is a lack of oxygen (hypoxia). Hypoxia is often experienced by athletes during training, especially during swimming training. Detection of gene expression can use qualitative and quantitative Polymerase chain reaction (PCR) methods. Each has advantages and disadvantages. Research on the expression of the HIF-1α gene in both humans and mice has been widely reported. However, the primary annealing temperature used varies, even with the same primer sequence. Therefore, it is necessary to optimize the primer annealing temperature before measuring gene expression. Annealing temperature optimization using the in-silico program showed annealing temperature at 52°C for the HIF-1alpa and 18s rRNA genes. Because the previous research used annealing temperature of 59ºC, the range of optimization temperature gradient was at 52ºC - 59ºC. Agarose gel electrophoresis results showed the HIF-1α gene product at 157bp and 18S rRNA gene 155bp. Optimum annealing temperature can be seen from the band thickness in the path of 53.6°C and 18S rRNA gene 55.8°C. Ct values for the HIF-1alpha gene are on average Ct to 25 and 18S rRNA genes at 10 ct. Based on good primer criteria, optimization of annealing temperature in silico, annealing temperature from the primer reference used, and the results of optimization of both genes in this study, the optimum annealing temperature recommended for further testing of the qPCR of all samples in this study series was 55.8 ° C.

Copyright

© 2020, the Authors. Published by Atlantis Press.

Open Access

This is an open access article distributed under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/).

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TY  - CONF
AU  - Siska Alicia Farma
AU  - Dezi Handayani
AU  - Dwi Hilda Putri
AU  - Argantos
AU  - Syahrastani
PY  - 2020
DA  - 2020/08/07
TI  - Optimization of Annealing Temperature of HIF-1 A and 18s rRNA in Blood of Swimming Athletes Using RT-PCR
BT  - Proceedings of the International Conference on Biology, Sciences and Education (ICoBioSE 2019)
PB  - Atlantis Press
SP  - 34
EP  - 38
SN  - 2468-5747
UR  - https://doi.org/10.2991/absr.k.200807.008
DO  - 10.2991/absr.k.200807.008
ID  - Farma2020
ER  -