Journal of Assisted Reproduction and Genetics, volume 35, issue 10, pages 1851-1860
Selection of stable expressed reference genes in native and vitrified/thawed human ovarian tissue for analysis by qRT-PCR and Western blot
Nikishin D A
1, 2
,
Filatov M A
1
,
Kiseleva M V
3
,
Bagaeva T S
1
,
Konduktorova V V
1
,
Khramova Y V
1
,
Malinova I V
3
,
Komarova E.V.
3
,
Publication type: Journal Article
Publication date: 2018-07-19
Quartile SCImago
Q2
Quartile WOS
Q2
Impact factor: 3.1
ISSN: 10580468
PubMed ID:
30027530
General Medicine
Genetics
Developmental Biology
Reproductive Medicine
Genetics (clinical)
Obstetrics and Gynecology
Abstract
PurposeTo select reference genes with stable messenger RNA (mRNA) expression for quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) analysis of vitrified/thawed human ovarian tissue and to evaluate in human ovarian tissue the levels of key proteins which are commonly used as reference proteins.MethodsPieces of ovarian tissue were obtained during laparoscopy from patients (n = 10, 24–36 years old) who suffered from types of cancer that does not affect reproductive system. Tissue strips from the intact group were immediately placed into liquid nitrogen. Tissue strips from the second group were successively placed into solutions with cryoprotective agents. Then, these strips were rapidly placed into liquid nitrogen. After thawing, ovarian tissue strips were cultured during 2 h in complete growth medium. Gene expression levels were measured using quantitative RT-PCR. Also, protein levels of three key reference genes were measured using Western blot. Statistical analysis of obtained data was performed by BestKeeper, NormFinder, and geNorm software utilities; correlation coefficients were also calculated.ResultsThe most suitable reference genes for qRT-PCR analysis of human cortical ovarian tissue after cryopreservation by vitrification are genes of ribosomal proteins RPL4, RPLP0, RPS18, and heat shock protein HSP90AB1. The protein levels of three commonly used reference genes (ACTB, GAPDH, and HSP90) were measured in two groups of samples of human ovarian tissue: intact and vitrified/thawed. The levels of ACTB, GAPDH, and HSP90 proteins were similar in native and vitrified/thawed samples.ConclusionSelection of suitable reference genes is the first aim of any research dedicated to the investigation of gene expression, because the interpretation of obtained results largely depends on selection of appropriate reference genes. Nowadays, there are many mathematical approaches allowing to select not only single reference gene but also a group of the most stably expressed reference genes. The use of mathematical models which take into account multiple reference genes will allow to obtain more accurate data on the expression of target genes.
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Nikishin D. A. et al. Selection of stable expressed reference genes in native and vitrified/thawed human ovarian tissue for analysis by qRT-PCR and Western blot // Journal of Assisted Reproduction and Genetics. 2018. Vol. 35. No. 10. pp. 1851-1860.
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Nikishin D. A., Filatov M. A., Kiseleva M. V., Bagaeva T. S., Konduktorova V. V., Khramova Y. V., Malinova I. V., Komarova E., Semenova S. L. Selection of stable expressed reference genes in native and vitrified/thawed human ovarian tissue for analysis by qRT-PCR and Western blot // Journal of Assisted Reproduction and Genetics. 2018. Vol. 35. No. 10. pp. 1851-1860.
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TY - JOUR
DO - 10.1007/s10815-018-1263-9
UR - https://doi.org/10.1007%2Fs10815-018-1263-9
TI - Selection of stable expressed reference genes in native and vitrified/thawed human ovarian tissue for analysis by qRT-PCR and Western blot
T2 - Journal of Assisted Reproduction and Genetics
AU - Nikishin, D A
AU - Filatov, M A
AU - Kiseleva, M V
AU - Bagaeva, T S
AU - Konduktorova, V V
AU - Khramova, Y V
AU - Malinova, I V
AU - Komarova, E.V.
AU - Semenova, S L
PY - 2018
DA - 2018/07/19 00:00:00
PB - Springer Nature
SP - 1851-1860
IS - 10
VL - 35
PMID - 30027530
SN - 1058-0468
ER -
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@article{2018_Nikishin,
author = {D A Nikishin and M A Filatov and M V Kiseleva and T S Bagaeva and V V Konduktorova and Y V Khramova and I V Malinova and E.V. Komarova and S L Semenova},
title = {Selection of stable expressed reference genes in native and vitrified/thawed human ovarian tissue for analysis by qRT-PCR and Western blot},
journal = {Journal of Assisted Reproduction and Genetics},
year = {2018},
volume = {35},
publisher = {Springer Nature},
month = {jul},
url = {https://doi.org/10.1007%2Fs10815-018-1263-9},
number = {10},
pages = {1851--1860},
doi = {10.1007/s10815-018-1263-9}
}
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Nikishin, D. A., et al. “Selection of stable expressed reference genes in native and vitrified/thawed human ovarian tissue for analysis by qRT-PCR and Western blot.” Journal of Assisted Reproduction and Genetics, vol. 35, no. 10, Jul. 2018, pp. 1851-1860. https://doi.org/10.1007%2Fs10815-018-1263-9.