Details

Autor(en) / Beteiligte

• Niu, Qiaoge
• Cao, Maosheng
• Chen, Shuxiong
• Zhao, Yun
• Chen, Lu
• Li, Chunjin
• Zhou, Xu

Titel

Effective and economical column-based method for RNA isolation from animal cells

Ist Teil von

• Biotechnology letters, 2019-09, Vol.41 (8-9), p.915-920

Ort / Verlag

Dordrecht: Springer Netherlands

Erscheinungsjahr

2019

Quelle

SpringerLink

Beschreibungen/Notizen

• Objectives To develop an efficient, economical, and low-toxicity method for the extraction of RNA from animal cells to meet a basic requirement of biological research: the isolation of high-quality RNA. Results Guanidine hydrochloride was used as a lysis buffer and Na-acetate was used as a wash buffer to extract RNA fragments from TM3 Leydig cells and ovarian granulosa cells efficiently. The functionality of the extracted RNA samples was verified through polymerase chain reaction (PCR) and real-time fluorescence quantitative PCR (RT-PCR). PCR results showed that the normal DNA column-based method could guarantee RNA integrity and could be used to amplify gene fragments successfully. RT-PCR analysis showed that the RNA samples isolated through the proposed method could be used to detect the expression levels of steroidogenic acute regulatory protein and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 mRNA in TM3 Leydig cells under induction by luteinizing hormone. The proposed method could be used to isolate RNA from mammalian cells and provided RNA yields of > 120 ng/5 × 10 6 cells. This method provided RNA with purities and yields that are sufficient for cDNA synthesis and PCR amplification in gene expression studies. Conclusions The proposed RNA extraction method has the advantages of low toxicity, safe handling, and low cost. Isolation can be completed in 20 min. The proposed method can be used to extract RNA from various animal cell samples and is worth promoting.