Trizol RNA ekstraksiyon metodu: inek plasentomu için oldukça etkili metod değerlendirmesi
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Date
2010-06-24
Authors
Shenavai, Sima
Journal Title
Journal ISSN
Volume Title
Publisher
Uludağ Üniversitesi
Abstract
Trizol gibi asit guanidium fenol kimyasalları, birçok numuneden yüksek-kalite total RNA izolasyonu için olanak sağlar. İnek plasentomundan (total, maternal ve fetal) ekonomik ve tekrar edilebilir bir metotla, yüksekkalite RNA izolasyonu yapabilmek için, total RNA 1 ml guanidin tiyosiyanat içeren asit solüsyonu, sodyum asetat, fenol ve kloroform ile santrifüj edilmiştir. Total RNA isopropanol ile çöktürülmüş ve hiçbir saflaştırma kiti kullanılmamıştır. İzolasyonlar iki farklı protokole göre yapılmıştır. İsopropanol fazına kadar çalışma (a) oda ısısında (20-22°C) ve (b) buz üzerinde (0-4°C) yapılmıştır. RNA kalitesi spektrofotometrik olarak, optik dansite (OD) ölçümü ile yapılmıştır (OD260 nm/OD280 nm > 1.7). RNA bütünlüğü agaroz jel elektroforezi ile kontrol edilmiştir. Elektroforezis sonuçlarına göre (a) protokolüne göre 28s, 18s ve 5s rRNA bantlar gözlenirken, (b) protokolüne göre çoğu zaman degrade RNA bantları gözlenmiştir. Total RNA konsantrasyonu (b) protokolüne göre belirgin ölçüde yüksek bulunurken, (a) protokolüne göre saflığı ve bütünlüğü çok iyi kaliteli RNA elde edilmiştir. Elde edilen total RNA’dan cDNA dönüşümü yapılarak PCR yapılmıştır. RT-PCR ürünleri (a) protokolüne göre izole edilen RNA’dan başarıyla elde edilmiştir. (a) protokolü, inek plasentomunun total, maternal ve fetal dokularından, hiçbir saflaştırma kiti kullanmadan, yüksek-kalite RNA izolasyonu için ekonomik ve tekrar edilebilir bir metod olduğunu göstermektedir.
Acid guanidium phenol preparations such as Trizol allow the reproducible isolation of high-quality total RNA from various sources. In order to establish an economical and reproducible method for the highquality RNA extraction from bovine total, maternal and fetal placenta, total RNA was isolated with 1 ml acidic solution containing guanidinium thiocyanate, sodium acetate, phenol and chloroform, followed by centrifugation. Total RNA was precipitated with isopropanol and no purification kit was used. The isolation was tested with two different protocols. Until isopropanol phase the isolation was carried out either a) at room temperatue (20-22°C) or b) on ice (0-4°C). The RNA quality was determined by spectrophotometry, optical density (OD) absorption ratio (OD260 nm/OD280 nm > 1.7). Integrity of the RNA was verified by agarose-gel electrophoresis. The results of electrophoresis showed three clear bands of 28s, 18s and 5s rRNA, respectively with protocol (a), whereas degraded RNA bands were mostly observed with protocol (b). Although the RNA concentration was significantly higher with protocol (b), the integrity and puririty were better with protocol (a). First strand cDNAs were amplified with extracted RNA using a kit, followed by basic PCR. The RT-PCR products were successfully derived from the extracted RNA. RNA extraction method with protocol (a) suggested an economical and reproducible method in obtaining high-quality RNA from fetal, maternal parts of bovine placenta with Trizol, without any purification method.
Acid guanidium phenol preparations such as Trizol allow the reproducible isolation of high-quality total RNA from various sources. In order to establish an economical and reproducible method for the highquality RNA extraction from bovine total, maternal and fetal placenta, total RNA was isolated with 1 ml acidic solution containing guanidinium thiocyanate, sodium acetate, phenol and chloroform, followed by centrifugation. Total RNA was precipitated with isopropanol and no purification kit was used. The isolation was tested with two different protocols. Until isopropanol phase the isolation was carried out either a) at room temperatue (20-22°C) or b) on ice (0-4°C). The RNA quality was determined by spectrophotometry, optical density (OD) absorption ratio (OD260 nm/OD280 nm > 1.7). Integrity of the RNA was verified by agarose-gel electrophoresis. The results of electrophoresis showed three clear bands of 28s, 18s and 5s rRNA, respectively with protocol (a), whereas degraded RNA bands were mostly observed with protocol (b). Although the RNA concentration was significantly higher with protocol (b), the integrity and puririty were better with protocol (a). First strand cDNAs were amplified with extracted RNA using a kit, followed by basic PCR. The RT-PCR products were successfully derived from the extracted RNA. RNA extraction method with protocol (a) suggested an economical and reproducible method in obtaining high-quality RNA from fetal, maternal parts of bovine placenta with Trizol, without any purification method.
Description
Keywords
Plasentom, İnek, Trizol, RNA, Placentom, Cattle
Citation
Özalp, G. R. vd. (2009). "Trizol RNA ekstraksiyon metodu: inek plasentomu için oldukça etkili metod değerlendirmesi". Uludağ Üniversitesi Veteriner Fakültesi Dergisi, 29(1), 1-6.