title: RNA isolation and quantitative PCR from HOPE- and formalin-fixed bovine lymph node tissues.
abstract: The use of ribonucleic acid (RNA) extracted from Hepes glutamic acid buffer-mediated organic solvent protection effect (HOPE)-fixed tissues in quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) is fairly novel. We compared qRT-PCR analysis of formalin- and HOPE-fixed, paraffin-embedded lymph node tissues from Mycobacterium bovis-infected cattle by extracting total RNA using a commercial kit (Ambion) and a Trizol method. RNA extracted from HOPE-fixed tissues showed comparable quantities between the commercial kit (82.7-107.9 microg/ml total RNA) and the Trizol method (87-161.1 microg/ml total RNA), displaying a high degree of integrity when analyzed by electrophoresis. RNA extracted from formalin-fixed tissues using the commercial kit produced similar concentrations (80.6-145.7 microg/ml total RNA) in comparison to the HOPE tissue; however, the integrity was compromised. Extraction of RNA from the formalin-fixed tissues using Trizol was unsuccessful. Following qRT-PCR for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), total RNA from HOPE-fixed tissues showed higher levels of target messenger ribonucleic acid (mRNA) (4.05 x 10(-2)pg/100 ng total RNA using the commercial kit and 6.45 x 10(-2)pg/100 ng total RNA using Trizol) in comparison to formalin-fixed tissues (5.69 x 10(-4)pg/100 ng total RNA). This could be attributed to RNA degradation by exposure to formalin fixation. In conclusion, the HOPE fixative proved to be a better source for RNA extraction from cattle lymph nodes and subsequent qRT-PCR.
authors.affiliation: School of Life Sciences, University of Hertfordshire, Hatfield, Herts AL10 9AB, UK.
authors.forename: Jaydene
authors.initials: J
authors.lastname: Witchell
authors.forename: Dhaval
authors.initials: D
authors.lastname: Varshney
authors.forename: Trusha
authors.initials: T
authors.lastname: Gajjar
authors.forename: Arun
authors.initials: A
authors.lastname: Wangoo
authors.forename: Madhu
authors.initials: M
authors.lastname: Goyal
mesh_terms: D000818:Animals; D002417:Cattle; D004587:Electrophoresis, Agar Gel; D005404:Fixatives; D005557:Formaldehyde / Q000737:chemistry*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006146:Guanidines / Q000737:chemistry; D006531:HEPES; D008198:Lymph Nodes / Q000201:enzymology / Q000382:microbiology; D009163:Mycobacterium bovis / Q000235:genetics; D010636:Phenols / Q000737:chemistry; D012313:RNA / Q000302:isolation & purification*; D020871:RNA Stability; D012329:RNA, Bacterial / Q000302:isolation & purification; D012333:RNA, Messenger / Q000302:isolation & purification; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D016707:Tissue Fixation / Q000379:methods* / Q000662:veterinary; D014380:Tuberculosis, Bovine / Q000175:diagnosis* / Q000235:genetics
chemical_list: D005404:Fixatives; D006146:Guanidines; D010636:Phenols; D012329:RNA, Bacterial; D012333:RNA, Messenger; C411644:trizol; D005557:Formaldehyde; D012313:RNA; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D006531:HEPES