Isolation of polysome-bound mRNA from solid tissues amenable for RT-PCR and profiling experiments.
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.
del Prete
M Julieta
MJ
Department of Immunology, Centro de Investigaciones Biológicas, Madrid, Spain.
Vernal
Rolando
R
Dolznig
Helmut
H
Müllner
Ernst W
EW
Garcia-Sanz
Jose A
JA
D053000:Analytic Sample Preparation Methods; D000818:Animals; D015152:Blotting, Northern; D002458:Cell Fractionation; D006493:Heparin / Q000737:chemistry; D006801:Humans; D018021:Lithium Chloride / Q000737:chemistry; D051379:Mice; D011132:Polyribosomes / Q000737:chemistry*; D012333:RNA, Messenger / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification*; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry; D018894:Reverse Transcriptase Inhibitors / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction
D012333:RNA, Messenger; D018894:Reverse Transcriptase Inhibitors; D006493:Heparin; D012194:RNA-Directed DNA Polymerase; D018021:Lithium Chloride