title: Assessment of different permeabilization methods of minimizing damage to the adherent cells for detection of intracellular RNA by flow cytometry.
abstract: 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).
  
  
  CONCLUSIONS
  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.
authors: Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran ; Student Research Committee, Shiraz University of Medical Sciences, Shiraz, Iran.
authors: Zahra
authors: Z
authors: Amidzadeh
authors: Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
authors: Abbas Behzad
authors: AB
authors: Behbahani
authors: Shiraz Institute for Cancer Research, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.
authors: Nasrollah
authors: N
authors: Erfani
authors: Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
authors: Sedigheh
authors: S
authors: Sharifzadeh
authors: Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
authors: Reza
authors: R
authors: Ranjbaran
authors: Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
authors: Leili
authors: L
authors: Moezi
authors: Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
authors: Farzaneh
authors: F
authors: Aboualizadeh
authors: Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
authors: Mohammad Ali
authors: MA
authors: Okhovat
authors: Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
authors: Parniyan
authors: P
authors: Alavi
authors: Shiraz Transplant Research center, Shiraz University of Medical Sciences, Shiraz, Iran.
authors: Negar
authors: N
authors: Azarpira
keywords: 18S rRNA; Flow cytometry; HeLa cells