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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

Gastric Biopsy sections with Wartin–Starry silver stain. (a) Mild infection (+) 0–10; (b) moderate (++) 11–30; and (c) severe infection (+++) >31 bacteria/field. The bacteria are indicated by the blue arrow.
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fig0010: Gastric Biopsy sections with Wartin–Starry silver stain. (a) Mild infection (+) 0–10; (b) moderate (++) 11–30; and (c) severe infection (+++) >31 bacteria/field. The bacteria are indicated by the blue arrow.

Mentions: The third adjacent block tape was placed on a polilysin-positive charged slide [1] and was processed in an Artisan Link Special Staining System for Wartin–Starry silver stain [2] (Fig. 2). This procedure allowed the use of high-resolution image microscopy (VIRTUAL; OLYMPUS) to identify the bacteria interacting with gastric epithelial cells. Thus, this third section was used to guide the selection of the area for LM in the HE stained samples [2] (Fig. 2).


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)

Gastric Biopsy sections with Wartin–Starry silver stain. (a) Mild infection (+) 0–10; (b) moderate (++) 11–30; and (c) severe infection (+++) >31 bacteria/field. The bacteria are indicated by the blue arrow.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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

fig0010: Gastric Biopsy sections with Wartin–Starry silver stain. (a) Mild infection (+) 0–10; (b) moderate (++) 11–30; and (c) severe infection (+++) >31 bacteria/field. The bacteria are indicated by the blue arrow.
Mentions: The third adjacent block tape was placed on a polilysin-positive charged slide [1] and was processed in an Artisan Link Special Staining System for Wartin–Starry silver stain [2] (Fig. 2). This procedure allowed the use of high-resolution image microscopy (VIRTUAL; OLYMPUS) to identify the bacteria interacting with gastric epithelial cells. Thus, this third section was used to guide the selection of the area for LM in the HE stained samples [2] (Fig. 2).

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