Comparison of Five Different RNA Isolation Methods from Equine Endometrium for Gene Transcription Analysis


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Kurar E., Atli M. O. , Guzeloglu A., Ozsensoy Y. , Semacan A.

KAFKAS UNIVERSITESI VETERINER FAKULTESI DERGISI, cilt.16, ss.851-855, 2010 (SCI İndekslerine Giren Dergi) identifier identifier

  • Cilt numarası: 16
  • Basım Tarihi: 2010
  • Dergi Adı: KAFKAS UNIVERSITESI VETERINER FAKULTESI DERGISI
  • Sayfa Sayıları: ss.851-855

Özet

In this study, five different isolation protocols to extract total RNA from biopsies of equine endometrium were compared in terms of quality and quantity of RNA samples with respect to downstream gene transcription analysis, such as Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Three phenol-chloroform based protocols (TRIzol, TRItidy, EZ-RNA) and two column based protocols (UltraClean (TM) and E.Z.N.A.(R)) that were commercially available were used. Each protocol yielded good quality total RNA and distinct 28S and 18S rRNA bands were observed in agarose gel electrophoreses. Amount of total RNA isolated was lower for EZ-RNA protocol. Column based protocols had RNA contaminated with great amount of genomic DNA, however, DNAse-I digestion was able to fully clean the DNA contamination from RNA in all the protocols used. Following cDNA synthesis and PCR, GAPDH, a housekeeping gene, bands were amplified from all the samples. In conclusion, all the protocols used extracted good quality but different amounts of total RNA and it is strongly recommended that RNA samples must undergo DNAse-I digestion before RT-PCR to eliminate gDNA contamination..