Isolation of high-quality RNA from white spruce tissue using a three-stage purification method and subsequent cloning of a transcript from the PR-10 gene family.

ABSTRACT

Isolation of PinmIII cDNA homologues from white spruce tissues required a rigorous RNA extraction protocol developed following assessment of three previously reported conifer RNA extraction protocols. Total RNA was extracted via several purification steps designed to minimize binding of phenolics to nucleic acids and was then subjected to caesium chloride ultra-centrifugation. This procedure produced consistently high-quality, intact RNA from both needles and roots with spectrophotometric ratios of approximately 2.0 for both 260/280 nm and 260/230 nm. Total RNA was obtained from the roots of cold-hardened white spruce seedlings for cDNA library construction. More than 2 million recombinant phage particles were generated from 5 microg of a poly(A)+RNA fraction, and ca. 1.3 million cDNA particles were amplified for storage. Approximately 500,000 primary recombinant clones were screened with an heterologous PinmIII cDNA sequence yielding a unique clone, picgl, that was very similar to members of the PR10 gene family.