BACKGROUND: Retrospective studies of archived human specimens, with known clinical follow-up, are used to identify predictive and prognostic molecular markers of disease. Due to biochemical differences, however, formalin-fixed paraffin embedded (FFPE) DNA and RNA have generally been extracted separately from either different tissue sections or from the same section by dividing the digested tissue. Our optimized co-extraction approach provides the option of collecting DNA, which would otherwise be discarded or degraded, for additional or subsequent studies because of the high importance and less availability of clinical FFPE specimen. METHODS: Coextraction of DNA and RNA from a single gastric cancer FFPE specimen was optimized by using TRIzol and purifying DNA from the lower aqueous and RNA from the upper organic phases. The protocol involves modification of incubation period for 30 min with proteinase K in glycin-tris-ethylenediamine tetra acetic acid buffer before adding TRIzol. RESULTS: All samples tested successfully performed semiquantitative gene expression by reverse transcriptase PCR. The quantity and quality of DNA from FFPE samples was high which resulted in successful PCR amplification. The isolated DNA also aided in detection of Helicobacter pylori by amplifying the ribosomal 16S gene in a multiplex PCR reaction along with cagA. CONCLUSION: These results show that the RNA/DNA isolated by this method can be used for easy clinical diagnosis of disease-related gene expression as well as mutation and pathogen detection from a homogenous population of tumor cells.
BACKGROUND: Retrospective studies of archived human specimens, with known clinical follow-up, are used to identify predictive and prognostic molecular markers of disease. Due to biochemical differences, however, formalin-fixed paraffin embedded (FFPE) DNA and RNA have generally been extracted separately from either different tissue sections or from the same section by dividing the digested tissue. Our optimized co-extraction approach provides the option of collecting DNA, which would otherwise be discarded or degraded, for additional or subsequent studies because of the high importance and less availability of clinical FFPE specimen. METHODS: Coextraction of DNA and RNA from a single gastric cancer FFPE specimen was optimized by using TRIzol and purifying DNA from the lower aqueous and RNA from the upper organic phases. The protocol involves modification of incubation period for 30 min with proteinase K in glycin-tris-ethylenediamine tetra acetic acid buffer before adding TRIzol. RESULTS: All samples tested successfully performed semiquantitative gene expression by reverse transcriptase PCR. The quantity and quality of DNA from FFPE samples was high which resulted in successful PCR amplification. The isolated DNA also aided in detection of Helicobacter pylori by amplifying the ribosomal 16S gene in a multiplex PCR reaction along with cagA. CONCLUSION: These results show that the RNA/DNA isolated by this method can be used for easy clinical diagnosis of disease-related gene expression as well as mutation and pathogen detection from a homogenous population of tumor cells.
Authors: Christina Thirlwell; Marianne Eymard; Andrew Feber; Andrew Teschendorff; Kerra Pearce; Matthias Lechner; Martin Widschwendter; Stephan Beck Journal: Methods Date: 2010-04-29 Impact factor: 3.608
Authors: Ravi Prakash Yadav; Souvik Ghatak; Payel Chakraborty; Freda Lalrohlui; Ravi Kannan; Rajeev Kumar; Jeremy L Pautu; John Zomingthanga; Saia Chenkual; Rajendra Muthukumaran; Nachimuthu Senthil Kumar Journal: Environ Sci Pollut Res Int Date: 2018-09-12 Impact factor: 4.223
Authors: Palak G Patel; Shamini Selvarajah; Suzanne Boursalie; Nathan E How; Joshua Ejdelman; Karl-Philippe Guerard; John M Bartlett; Jacques Lapointe; Paul C Park; John B A Okello; David M Berman Journal: J Vis Exp Date: 2016-08-21 Impact factor: 1.355