Limits...
Improved method for extraction and detection of Helicobacter pylori DNA in formalin-fixed paraffin embedded gastric biopsies using laser micro-dissection.

Loayza MF, Villavicencio FX, Santander SC, Baldeón M, Ponce LK, Salvador I, Vivar Díaz N - MethodsX (2014)

Bottom Line: Few articles have described the use of LM to select and detect H. pylori genome from formalin-fixed paraffin embedded gastric tissue [2].The mean of DNA concentration obtained from 25 LM cut sections was 1.94± 0 .16 ng/μL, and it was efficiently amplified with qPCR in a Bio Rad iCycler instrument.The LM can improve the sample selection and DNA extraction for molecular analysis of H. pylori associated with human gastric epithelium.

View Article: PubMed Central - PubMed

Affiliation: Universidad de las Fuerzas Armadas ESPE, P.O. Box 171-5-231B, Av. General Rumiñahui s/n, Sangolquí, Ecuador ; Hospital Carlos Andrade Marín, P.O. Box 170411, Av. 18 de Septiembre s/n y Ayacucho, Quito, Ecuador.

ABSTRACT
To assess the molecular events exerted by Helicobacter pylori interacting directly with gastric epithelial cells, an improved procedure for microbial DNA isolation from stained hematoxilin-eosin gastric biopsies was developed based on laser micro-dissection (LM) [1]. Few articles have described the use of LM to select and detect H. pylori genome from formalin-fixed paraffin embedded gastric tissue [2]. To improve the yield and quality of DNA isolated from H. pylori contacting intestinal epithelial cells, the following conditions were established after modification of the QIAamp DNA Micro kit. •Use of at least 25 cut sections of 10-20 μm of diameter and 3 μm thick with more than 10 bacteria in each cut.•Lysis with 30 μL of tissue lysis buffer and 20 μL of proteinase K (PK) with the tube in an upside-down position.•The use of thin purification columns with 35 μL of elution buffer. The mean of DNA concentration obtained from 25 LM cut sections was 1.94± 0 .16 ng/μL, and it was efficiently amplified with qPCR in a Bio Rad iCycler instrument. The LM can improve the sample selection and DNA extraction for molecular analysis of H. pylori associated with human gastric epithelium.

No MeSH data available.


Related in: MedlinePlus

Point A shows the DNA concentration that was isolated from 5 tape cuts from FFPE gastric tissue block (each tape was 3 μm thick). Points B–E along the X-axis represent the number of LM cut sections (between 5 and 25), with more than 10 bacteria added to the gastric surface. The cut sections were added to the cap of a 0.2 mL tube and were used to perform DNA isolation assay. The Y-axis shows the DNA concentration, reported in ng/μL.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4487329&req=5

fig0020: Point A shows the DNA concentration that was isolated from 5 tape cuts from FFPE gastric tissue block (each tape was 3 μm thick). Points B–E along the X-axis represent the number of LM cut sections (between 5 and 25), with more than 10 bacteria added to the gastric surface. The cut sections were added to the cap of a 0.2 mL tube and were used to perform DNA isolation assay. The Y-axis shows the DNA concentration, reported in ng/μL.

Mentions: Data showed that the greatest number of tissue sections obtained with LM yielded greatest concentrations of DNA. The average DNA concentration obtained from 25 LM cut sections was 1.94 ± 0.16 ng/μL. The DNA concentration obtained from LM samples was 10-fold less than the concentration obtained following conventional extraction from FFPE gastric biopsies (Fig. 4). Fig. 5 indicates the consistency of the method to isolate microbial DNA. Although the final concentration of DNA isolated by this method is less than the amount of DNA isolated by conventional strategies, the obtained nucleic acid is of bacterial origin.


Improved method for extraction and detection of Helicobacter pylori DNA in formalin-fixed paraffin embedded gastric biopsies using laser micro-dissection.

Loayza MF, Villavicencio FX, Santander SC, Baldeón M, Ponce LK, Salvador I, Vivar Díaz N - MethodsX (2014)

Point A shows the DNA concentration that was isolated from 5 tape cuts from FFPE gastric tissue block (each tape was 3 μm thick). Points B–E along the X-axis represent the number of LM cut sections (between 5 and 25), with more than 10 bacteria added to the gastric surface. The cut sections were added to the cap of a 0.2 mL tube and were used to perform DNA isolation assay. The Y-axis shows the DNA concentration, reported in ng/μL.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4487329&req=5

fig0020: Point A shows the DNA concentration that was isolated from 5 tape cuts from FFPE gastric tissue block (each tape was 3 μm thick). Points B–E along the X-axis represent the number of LM cut sections (between 5 and 25), with more than 10 bacteria added to the gastric surface. The cut sections were added to the cap of a 0.2 mL tube and were used to perform DNA isolation assay. The Y-axis shows the DNA concentration, reported in ng/μL.
Mentions: Data showed that the greatest number of tissue sections obtained with LM yielded greatest concentrations of DNA. The average DNA concentration obtained from 25 LM cut sections was 1.94 ± 0.16 ng/μL. The DNA concentration obtained from LM samples was 10-fold less than the concentration obtained following conventional extraction from FFPE gastric biopsies (Fig. 4). Fig. 5 indicates the consistency of the method to isolate microbial DNA. Although the final concentration of DNA isolated by this method is less than the amount of DNA isolated by conventional strategies, the obtained nucleic acid is of bacterial origin.

Bottom Line: Few articles have described the use of LM to select and detect H. pylori genome from formalin-fixed paraffin embedded gastric tissue [2].The mean of DNA concentration obtained from 25 LM cut sections was 1.94± 0 .16 ng/μL, and it was efficiently amplified with qPCR in a Bio Rad iCycler instrument.The LM can improve the sample selection and DNA extraction for molecular analysis of H. pylori associated with human gastric epithelium.

View Article: PubMed Central - PubMed

Affiliation: Universidad de las Fuerzas Armadas ESPE, P.O. Box 171-5-231B, Av. General Rumiñahui s/n, Sangolquí, Ecuador ; Hospital Carlos Andrade Marín, P.O. Box 170411, Av. 18 de Septiembre s/n y Ayacucho, Quito, Ecuador.

ABSTRACT
To assess the molecular events exerted by Helicobacter pylori interacting directly with gastric epithelial cells, an improved procedure for microbial DNA isolation from stained hematoxilin-eosin gastric biopsies was developed based on laser micro-dissection (LM) [1]. Few articles have described the use of LM to select and detect H. pylori genome from formalin-fixed paraffin embedded gastric tissue [2]. To improve the yield and quality of DNA isolated from H. pylori contacting intestinal epithelial cells, the following conditions were established after modification of the QIAamp DNA Micro kit. •Use of at least 25 cut sections of 10-20 μm of diameter and 3 μm thick with more than 10 bacteria in each cut.•Lysis with 30 μL of tissue lysis buffer and 20 μL of proteinase K (PK) with the tube in an upside-down position.•The use of thin purification columns with 35 μL of elution buffer. The mean of DNA concentration obtained from 25 LM cut sections was 1.94± 0 .16 ng/μL, and it was efficiently amplified with qPCR in a Bio Rad iCycler instrument. The LM can improve the sample selection and DNA extraction for molecular analysis of H. pylori associated with human gastric epithelium.

No MeSH data available.


Related in: MedlinePlus