Simultaneous extraction of total RNA and peptides from tissues: application to tachykinins.
Methods for extraction and isolation of intact RNA are often laborious, time consuming, and preclude the direct analysis of peptides. Similarly, the conditions for extraction and isolation of peptides are unsuitable for the isolation of intact RNA. Thus, to study changes in the levels of neuropeptides and gene expression of the corresponding mRNAs, separate procedures are required. A simple and rapid method for the simultaneous extraction of RNA and peptides from tissues is described. RNA and peptides are extracted with guanidinium isothiocyanate, followed by delipidation, and peptides are isolated by a simple solid-phase extraction procedure. RNA is isolated by differentially partitioning DNA into an organic phase, followed by precipitation with ethanol. The RNA and peptides isolated by this method are of high yield and quality. Furthermore, this method for RNA isolation is successful and efficient, even with tissues that proved recalcitrant with other procedures, and allows the simultaneous processing of multiple samples. We describe the successful application of this procedure for measuring tachykinins and the corresponding preprotachykinin A mRNA from tissues. Extraction of neuropeptide K, a 36-mer tachykinin, was dramatically more efficient with the present method than other methods in common use.
Too
H P
HP
Department of Biochemistry, National University of Singapore.
Maggio
J E
JE
D000818:Animals; D001923:Brain Chemistry; D002851:Chromatography, High Pressure Liquid; D005260:Female; D007668:Kidney / Q000737:chemistry; D008099:Liver / Q000737:chemistry; D008297:Male; D008722:Methods; D010455:Peptides / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D012313:RNA / Q000032:analysis* / Q000302:isolation & purification; D051381:Rats; D017207:Rats, Sprague-Dawley; D013116:Spinal Cord / Q000737:chemistry / Q000276:immunology; D013373:Substance P / Q000737:chemistry / Q000276:immunology; D015320:Tachykinins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification
D010455:Peptides; D015320:Tachykinins; D013373:Substance P; D012313:RNA