A modified protocol for RNA isolation from high polysaccharide containing Cupressus arizonica pollen. Applications for RT-PCR and phage display library construction.
RNA isolation is the first step in the study of gene expression and recombinant protein production. However, the isolation of high quantity and high-quality RNA from tissues containing large amounts of polysaccharides has proven to be a difficult process. Cupressus arizonica pollen, in addition to containing high polysaccharide levels, is a challenging starting material for RNA isolation due to the roughness of the pollen grain's walls. Here, we describe an improved technique for RNA isolation from C. arizonica pollen grains. The protocol includes a special disruption and homogenization process as well as a two-step modified RNA isolation technique which consists of an acid phenol extraction followed by a final cleanup using a commercial kit. Resulting RNA proved to be free of contaminants as determined by UV spectrophotometry. The quality of the RNA was analyzed on a bioanalyzer and showed visible 25S and 18S bands. This RNA was successfully used in downstream applications such as RT-PCR and phage display library construction.
Pico de Coaña
Yago
Y
Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Spain. ypico@cbm.uam.es
Parody
Nuria
N
Fernández-Caldas
Enrique
E
Alonso
Carlos
C
D029781:Cupressus / Q000235:genetics / Q000378:metabolism*; D015723:Gene Library; D011058:Pollen / Q000235:genetics / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism*; D018749:RNA, Plant / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013056:Spectrophotometry, Ultraviolet
D011134:Polysaccharides; D018749:RNA, Plant