RESUMEN
Background: DNA isolation procedure can significantly influence the quantification of DNA by real time PCR specially when cell free DNA [cfDNA] is the subject. To assess the extraction efficiency, linearity of the extraction yield, presence of co-purified inhibitors and to avoid problems with fragment size relevant to cfDNA, development of appropriate External DNA Control [EDC] is challenging. Using non-human chimeric nucleotide sequences, an EDC was developed for standardization of qPCR for monitoring stability of cfDNA concentration in blood samples over time
Methods: A DNA fragment of 167 bp chimeric sequence of parvovirus B19 and pBHA designated as EDC fragment was designed. To determine the impact of different factors during DNA extraction processing on quantification of cfDNA, blood samples were collected from normal subjects and divided into aliquots with and without specific treatment. In time intervals, the plasma samples were isolated. The amplicon of 167 bp EDC fragment in final concentration of 1.1 pg/ 500 microl was added to each plasma sample and total DNA was extracted by an in house method. Relative and absolute quantification real time PCR was performed to quantify both EDC fragment and cfDNA in extracted samples
Results: Comparison of real time PCR threshold cycle [Ct] for cfDNA fragment in tubes with and without specific treatment indicated a decrease in untreated tubes. In contrast, the threshold cycle was constant for EDC fragment in treated and untreated tubes, indicating the difference in Ct values of the cfDNA is because of specific treatments that were made on them
Conclusions: Spiking of DNA fragment size relevant to cfDNA into the plasma sample can be useful to minimize the bias due to sample preparation and extraction processing. Therefore, it is highly recommended that standard external DNA control be employed for the extraction and quantification of cfDNA for accurate data analysis
RESUMEN
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. 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. 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]. 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