An improved method for mRNA isolation and characterization of in vitro translation products by Western blotting.
A method is described for isolation of messenger RNA (mRNA) from a rather intractable tissue source, calf stomach. The use of additional RNase inhibitors, vanadyl ribonucleoside complexes and proteinase K, which are used in conjunction with the guanidine thiocyanate/CsCl ultracentrifugation procedure traditionally employed for isolation of mRNA, is described. These modifications make the procedure universally applicable to a wide variety of tissues and cell types. The validity of the procedure is demonstrated by isolation of biologically active full-length preprochymosin mRNA. The integrity of the mRNA is measured by in vitro translation, Northern blot analysis, Southern blot analysis of preprochymosin cDNA using synthetic oligodeoxynucleotide probes and immunospecific identification of in vitro translation products using a modification of the Western blot which is described in this report.
D000818:Animals; D002417:Cattle; D011232:Chemical Precipitation; D012085:Chymosin / Q000235:genetics; D004792:Enzyme Precursors / Q000235:genetics; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification*; D012260:Ribonucleases / Q000037:antagonists & inhibitors; D013270:Stomach / Q000032:analysis
D004792:Enzyme Precursors; D012333:RNA, Messenger; D012260:Ribonucleases; C034675:preprorennin; D012085:Chymosin