A convenient and efficient protocol for isolating high-quality RNA from latex of Hevea brasiliensis (para rubber tree).
Isolating high-quality RNA from latex of H. brasiliensis is a prerequisite to elucidating the molecular mechanisms of rubber biosynthesis and its regulation. Here, an improved protocol was developed for latex collection, transportation, storage, and RNA isolation. Compared with existing ones, our protocol eliminated liquid nitrogen for latex collection and subsequent low-temperature (-70 degrees C) condition for latex storage, making it more convenient and feasible when latex was collected in remote sampling sites, and latex storage and RNA isolation were conducted in poorly-equipped laboratories. Different methods (UV absorbance scans, denaturing gel electrophoresis, autoradiograph monitoring of cDNA synthesis) were used to confirm the high quality of the RNA prepared with this protocol, whose usefulness was further verified by several practical applications, including construction of one high-quality cDNA library, cloning of the full-length cDNAs of 3 novel Hevea sucrose transporter genes, and semi-quantitative RT-PCR analysis of two rubber-biosynthesis essential genes and one sucrose transporter gene.
Tang
Chaorong
C
Key Lab of Ministry of Agriculture for Tropical Crops Physiology, Rubber Research Institute, Chinese Academy of Tropical Agricultural Sciences, Danzhou, Hainan 571737, China. chaorongtang@126.com
Qi
Jiyan
J
Li
Heping
H
Zhang
Cunliang
C
Wang
Yuekun
Y
D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D015723:Gene Library; D017343:Genes, Plant; D028482:Hevea / Q000737:chemistry* / Q000235:genetics; D007840:Latex / Q000737:chemistry*; D026901:Membrane Transport Proteins / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics; D018749:RNA, Plant / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D048348:Reverse Transcription
D017931:DNA Primers; D018076:DNA, Complementary; D018744:DNA, Plant; D007840:Latex; D026901:Membrane Transport Proteins; D010940:Plant Proteins; D018749:RNA, Plant; C078729:sucrose transport protein, plant