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Avicenna Journal of Medical Biotechnology، جلد ۶، شماره ۱، صفحات ۳۸-۴۶
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| عنوان فارسی |
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| چکیده فارسی مقاله |
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| کلیدواژه‌های فارسی مقاله |
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| عنوان انگلیسی |
Assessment of Different Permeabilization Methods of Minimizing Damage to the Adherent Cells for Detection of Intracellular RNA by Flow Cytometry |
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| چکیده انگلیسی مقاله |
Background: Various fixation and permeabilization techniques have been developed for detection of intracellular antigens by flow cytometry; however, there are few studies using flow cytometry to detect the frequency of intracellular nucleic acids, particularly RNA. We tested six different permeabilization methods in order to gain access to a high quality method with minimal damage to intracellular components focusing on 18S rRNA in HeLa cells. Methods: HeLa cells were fixed in 2% paraformaldehyde. A variety of detergents and enzymes including saponin, TritonX-100, Tween-20, NP40, Proteinase K, and streptolysin O were used to optimize a protocol of permeabilization for the flow cytometric enumeration of intracellular 18S rRNA. Treated cells were subjected to standard protocol of flow cytometric in situ hybridization in the presence of FITC-labeled sense and antisense probes to detect 18S ribosomal RNAs. Samples were then analyzed on a FACSCalibur flow cytometer. To evaluate cell morphology, following hybridization the cells were fixed on glass slide, covered with DAPI, and evaluated on a fluorescent microscope with appropriate filter sets. Results: In comparison with other methods, maximum cell frequency in percentage and fluorescent intensity (M1=2.1%, M2=97.9%) were obtained when the cells were treated with 0.2% Tween-20 and incubated for 30 min (p=0.001). Conclusion: Our study indicated that the highest levels of mean fluorescence could be obtained when the cells were treated with Tween-20. However, it should be taken into consideration that for a successful flow cytometric result, other interfering factors such as hybridization conditions should also be optimized. |
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| کلیدواژه‌های انگلیسی مقاله |
18S rRNA, Flow cytometry, HeLa cells |
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| نویسندگان مقاله |
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| نشانی اینترنتی |
http://www.ajmb.org/En/Article.aspx?id=140 |
| فایل مقاله |
اشکال در دسترسی به فایل - ./files/site1/rds_journals/133/article-133-376124.pdf |
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| زبان مقاله منتشر شده |
en |
| موضوعات مقاله منتشر شده |
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