Isolation of polysome-bound mRNA from solid tissues amenable for RT-PCR and profiling experiments

Abstract

Using cell lines and primary cells, it has been shown that translation control plays a key role regulating gene expression during physiological and pathological conditions. The relevance of this type of regulation in vivo (tissues, organs) remains to be elucidated, due to the lack of an efficient method for polysome-bound fractionation of solid tissue RNA samples. A simple and efficient method is described, in which tissue samples were pulverized in liquid nitrogen and lysed with NP40-lysis buffer in the presence of the RNAse inhibitors RNAsin and vanadyl-ribonucleoside complex. After cell lysis, the cytoplasmic extract was loaded into sucrose gradients, fractionated, and RNA prepared from each fraction. The obtained RNA was reverse transcribed with a low efficiency, a problem that was overcome by purifying polyA + RNA. Aiming to use small quantities of solid tissue samples (10-20 mg/sample), polyA + RNA purification was discarded, and the different components were individually screened for a negative effect on reverse transcription. The polysaccharide heparin, which is present as a nonspecific RNAse inhibitor, inhibits reverse transcriptase activity, and must be removed from RNA samples for an efficient reaction. Heparin was successfully removed by precipitation of the RNA with lithium chloride, as demonstrated by the reversal of the inhibition on RT-PCR reactions. In summary, we present a reliable method allowing us to prepare high-quality polysome-bound mRNA from small quantities of liquid-nitrogen-frozen solid tissue samples from both human and mouse origin, amenable for Northern blotting, RT-PCR reactions, and expression profiling analyses.

Alternative RNA Splicing Produces Multiple Forms of c-Myb with Unique Transcriptional Activities

“…Transcripts associated with polyribosomes were analyzed as described previously (9). Briefly, K562 cell cytoplasmic fractions were layered onto 40 to 60% Nyzode (Sigma, St. Louis, MO) gradients containing either cycloheximide (for ribosome clamping) or EDTA (for ribosome release).…”

PU.1 expression is modulated by the balance of functional sense and antisense RNAs regulated by a shared cis-regulatory element

“…Polysomes were isolated from cytoplasmic extracts by sucrose sedimentation. We used methods derived from the protocols developed for nuclei isolation by Blobel and Potter (1966), for cytoplasmic RNA prepara-tion by del Prete et al (2007), and for polysome isolation by Fennoy et al (1997) with minor modifications.…”

<em>In vivo</em> Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

“…This is especially the case for in vivo CNS samples, where the amount of material is often a limitation and fatty tissue components hinder isolation of translating mRNAs. Most published fractionation protocols deal with yeast or mammalian cell lines, and there are established protocols for the brain 12,13,14 . In contrast, there are barely any publications describing fractionation of the spinal cords, and previous protocols require spinal cord from a large number of animals to be pooled 15 .…”