RNA ISOLATION AND DETECTION OF CELLULAR RNA QUANTITY OF SPERMATOZOA AND EMBRYOS PRIOR TO GENE EXPRESSION ANALYSES

Year 2018, Volume: 81 Issue: 4, 119 - 126, 01.12.2018

Bilge Özsait Selçuk , Neslihan Çoban , Dilek Sever-kaya Sibel Bulgurcuoğlu-kuran Selva Türkölmez Özlem Dural

Abstract

DOI: 10.26650/IUITFD.416666

Objective: Biological samples that are analyzed
in reproductive biology are generally rare, difficult to obtain, and a
nonreplicable group of cells. Furthermore, investigating low numbers of cells
requires modifications to the routine methods used in genetic analyses. The aim
of our study was to improve RNA isolation methods for obtaining a sufficient
amount of total RNA from spermatozoa and embryo samples for downstream gene
expression analyses.

Materials and Methods: Excess spermatozoa
samples (that had been prepared in the scope of assisted reproduction
treatment) were purified of any contaminant cells and then frozen.
Preimplantation stage embryos that had not been selected for embryo transfer
were frozen in a phosphate-buffered saline–polyvinyl alcohol (PBS–PVA)
solution. Modifications were done to obtain the optimal amount of total RNA
from the spermatozoa and embryos, and subsequently both the quality and the
quantity of the total RNA samples were assessed. Because of the selective
degradation of 28S RNA samples in the spermatozoa, the total RNA quality was
evaluated by gene amplification using quantitative real-time polymerase chain
reaction (qRT-PCR) in addition to Bioanalyzer analysis.

Results: Following the modifications to the
isolation technique, a sufficient quality and quantity of total RNA were
obtained from the spermatozoa and embryo samples, which could be used in
downstream gene expression analyses. Furthermore, the amount of cellular total
RNAs was consistent to that reported in previous studies.

Conclusion: Results obtained from these
experiments - conducted as a preliminary work for further transcript profiling
studies - indicate that the modifications used in the RNA isolation techniques
were effective.

Keywords

Spermatozoon, preimplantation stage embryo

SPERMATOZOA VE EMBRİYOLARIN GEN EKSPRESYON ANALİZİ ÖNCESİNDE RNA İZOLASYONU VE HÜCRESEL RNA MİKTARLARININ BELİRLENMESİ

Abstract

DOI: 10.26650/IUITFD.416666

Amaç: Üreme biyolojisi araştırmalarında
kullanılan örnekler genellikle, zor ulaşılabilir, nadir ve tekrarı olmayan
hücre gruplarıdır. Diğer yandan, az sayıda hücre ile çalışıldığından dolayı,
genetik analizlerde standart olarak kullanılan yöntemlerde modifikasyonlara
ihtiyaç duyulmaktadır. Bu çalışmada amacımız, gen ekspresyonu araştırmalarında
kullanılabilecek optimal düzeydeki total RNA’nın elde edilebilmesi amacı ile
spermatozoa ve embriyo örneklerinden RNA izolasyon yönteminin
geliştirilmesidir.

Gereç ve Yöntem: Yardımla üreme tedavisi
kapsamında kullanılmak amacı ile hazırlanan ve fertilizasyon işlemi
gerçekleştirildikten sonra geriye kalan spermatozoa örneği diğer kontaminant
hücrelerden saflaştırılarak dondurulmuştur. Diğer yandan, embriyo transferinden
sonra geriye kalan preimplantasyon dönem embriyolar fosfat tamponlu tuz –
polivinil alkol (PBS-PVA) karışımı içerisinde dondurularak saklanmıştır.
Spermatozoa ve embriyolardan optimal düzeyde total RNA elde edilmesi amacı ile
çeşitli optimizasyonlar yapılmıştır. Elde edilen total RNA’lar kaliteleri ve
miktarları açısından değerlendirilmiştir. Spermatozoonda 28S ribozomal RNA
(rRNA)’nın seçimli olarak degrade olmasından dolayı total RNA kalitesi,
Bioanalyzer cihazı ile gerçekleştirilen analize ek olarak gerçek zamanlı
kantitatif PCR ile gen amplifikasyonu yapılarak da kontrol edilmiştir.

Bulgular: İzolasyonda yapılan
uyarlamalardan sonra, takip eden gen ekspresyonu çalışmalarında
kullanılabilecek kalitede ve miktarda total RNA elde edilmiştir. Ek olarak,
örneklerden elde edilen hücresel total RNA miktarlarının önceki çalışmalar ile
uyumlu olduğu belirlenmiştir.

Sonuç: Daha sonra yapılması planlanan
transkript profilleme araştırmaları için ön bir çalışma olarak gerçekleştirilen
bu deneylerin sonucunda elde edilen bulgular, RNA izolasyon protokollerinde
yapılan uyarlamaların etkin olduğunu göstermektedir. 

Keywords

spermatozoon, preimplantasyon dönem embriyo

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