title: Formamide-based RNA isolation at above zero temperatures from high starch cassava tubers.
abstract: BACKGROUND
  Cassava (Manihot esculenta) tubers are a main source of carbohydrate for a large percentage of people in the tropics. However, obtaining RNA from such high starch tubers proves problematic because gelation occurs during standard isolation procedures. In preliminary studies, formamide was used to homogenise tubers at room temperature and no gelation was observed.
  
  
  OBJECTIVE
  To develop a simple, reproducible formamide-based procedure for RNA isolation from high starch tissues without the initial use of liquid nitrogen or lyophilisation. A second objective was to determine the impact of storage of formamide homogenates at 4°C on RNA integrity.
  
  
  METHODS
  Tubers were homogenised in 100% formamide at room temperature and stored for 1, 4 or 7 days at 4°C. Homogenates were centrifuged at 14000 × g for 10 min and RNA recovered from the supernatants by isopropanol precipitation followed by dissolution in guanidinium buffer. Protein was removed by standard phenol-chloroform-isoamyl alcohol extraction and RNA recovered by isopropanol precipitation. For immediate RNA isolation, formamide homogenates can be processed without incubation at 4°C.
  
  
  RESULTS
  In formamide homogenates a non-gelatinous white pellet was observed which was identified as starch by Lugol's staining. Intact RNA was observed by agarose gel electrophoresis. The RNA was successfully used in downstream RT-PCR reactions.
  
  
  CONCLUSIONS
  Formamide maintained the integrity of the RNA during tissue processing at 25 ± 2°C and storage at 4°C. This room temperature formamide-based RNA isolation procedure met the criteria of simplicity, low-cost, reproducibility and addressed gelation problems associated with high starch tissues.
authors: Department of Life Sciences, The University of The West Indies, St Augustine, Trinidad and Tobago. Bowrin@sta.uwi.edu
authors: Valerie
authors: V
authors: Bowrin
authors: Judy
authors: J
authors: Rouse-Miller
authors: Fedora
authors: F
authors: Sutton
authors: Grace
authors: G
authors: Sirju-Charran
mesh_terms: D001671:Biochemistry / Q000379:methods*; D005559:Formamides / Q000737:chemistry; D002365:Manihot / Q000235:genetics*; D035281:Plant Tubers / Q000235:genetics*; D018749:RNA, Plant / Q000302:isolation & purification*; D015203:Reproducibility of Results; D013213:Starch / Q000235:genetics*; D013696:Temperature
chemical_list: D005559:Formamides; D018749:RNA, Plant; C031066:formamide; D013213:Starch