A method for obtaining high quality RNA from paraffin sections of plant tissues by laser microdissection.
Laser microdissection (LM) combined with microarray analysis or next-generation sequencing of cDNA is a powerful tool for understanding molecular events in individual cell types of plants as well as animals. Obtaining high quality RNA is essential for this approach. For plant tissues, paraffin-embedded sections better preserve cell structure than do frozen sections. However, the conventional method for preparing paraffin sections is a lengthy process involving embedding the tissue and floating and drying the sections, during which time RNA degradation occurs. Here, we describe a method for preparing serial sections that greatly reduces RNA degradation: we reduced (1) the embedding time from 4-6 days to about 5 h by using a recently developed microwave method; (2) the time of floating sections from ~10 min to less than 5 min, (3) the drying time from ~12 to 1 h; and (4) the drying temperature from 42 to 4°C. With this method, we were able to isolate higher integrity RNA from many kinds of plant tissues than is typically obtained by the conventional paraffin preparation method. The improvement in RNA quality and yield removes a major obstacle to the widespread use of LM with high-throughput technologies for plants.
Takahashi
Hirokazu
H
Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo, 113-8657, Japan.
Kamakura
Hisae
H
Sato
Yutaka
Y
Shiono
Katsuhiro
K
Abiko
Tomomi
T
Tsutsumi
Nobuhiro
N
Nagamura
Yoshiaki
Y
Nishizawa
Naoko K
NK
Nakazono
Mikio
M
D007834:Lasers; D042282:Microdissection / Q000379:methods*; D008872:Microwaves; D009928:Organ Specificity; D012275:Oryza / Q000166:cytology / Q000235:genetics*; D016612:Paraffin Embedding / Q000379:methods*; D018749:RNA, Plant / Q000302:isolation & purification* / Q000592:standards*; D013997:Time Factors
D018749:RNA, Plant