Isolation of total RNA from pollens.
Isolation of total RNA from plant materials has been difficult, due to the presence of complex organic substances and the associated pigmentation. In fact, there is a dearth of standardized protocols for isolating total RNA from pollens. To find a simple and reliable method for isolating total RNA from pollen, four methods, viz. phenol/SDS (PS), guanidine HCl (GH), tri-reagent (TR), and modified SDS-betaME (SB) were tested with fresh pollen of Ricinus communis (procured at -70 degrees C) and pollen dried at 30-37 degrees C. The quality and quantity of RNA was superior for the material processed at -70 degrees C. SB gave the highest RNA yield (2.35 mg/g, OD260/280 >2.0), compared to other methods. The results obtained by the SB method were found to be comparable with the widely used tri-reagent method. This was validated with other pollens of Imperata cylindrica and Xanthium strumarium. The yield obtained from graded amounts of pollen was consistent with SB, compared to the TR method. The RNA isolated by SB gave good quality mRNA for synthesizing cDNA. The SDS-betaME method is simple, efficient, and uses less expensive reagents. Hence, we recommend the modified SDS-betaME method for isolating total RNA from pollens.
Bijli
K M
KM
Centre for Biochemical Technology, Delhi University Campus, India.
Singh
B P
BP
Sridhara
S
S
Arora
N
N
D019791:Guanidine / Q000737:chemistry; D006146:Guanidines / Q000737:chemistry; D008623:Mercaptoethanol / Q000737:chemistry; D010636:Phenols / Q000737:chemistry; D011058:Pollen / Q000737:chemistry*; D018749:RNA, Plant / Q000302:isolation & purification*; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013861:Thiocyanates / Q000737:chemistry
D006146:Guanidines; D010636:Phenols; D018749:RNA, Plant; D013861:Thiocyanates; D012967:Sodium Dodecyl Sulfate; C054436:guanidine thiocyanate; D008623:Mercaptoethanol; D019791:Guanidine