RNA is highly labile and degrades quickly if stored under improper conditions. RNA molecules are susceptible to a variety of secondary metabolites, and enzymatic degradation by ribonucleases RNases , causing problems during extraction [ 5 ]. The separation of exfoliated cells from the brush is important for RNA extraction. Because most RNA stabilization reagents are sticky, and the tube is rigid, separation of the exfoliated cells from the brush is difficult.
However, shipping and storage of frozen RNA is expensive, requires special handling and Ultra-Low-Temperature freezers, and is time sensitive. To the best of our knowledge, comparison of mRNA extraction methods for molecular analysis of cervico-vaginal cytology samples has not been reported. Therefore, the aim of this study was 1 to compare methods of RNA extraction of cervico-vaginal cytology as they relate to the type of tubes, preservative solutions, and storage temperature and 2 to establish the best technique for molecular tests such as real-time reverse transcriptase reaction qRT PCR or RNA-seq at room temperature.
After obtaining approval from the Institutional Review Board of our institution, and informed written consent from the subjects, cervico-vaginal smears were collected using pap brush-lines Bion, Guri, Korea in patients with HPV positive cervical intraepithelial neoplasia or cervical cancer.
The brush was placed into the cervix and rotated degrees in a clockwise direction. The samples were transported in DNase, RNase and pyrogenic free tubes. To obtain the cells from the cytobrush, the cytobrush was shaken in rigid tube, or the tube and brush were squeezed in a soft tube Fig.
Detailed steps to separate exfoliated cervico-vaginal cells from the brush using soft elastic tubes were as follows: First, the brush was inserted into the soft tube Fig. Second, the upper portion of the soft tube and cytobrush were grasped by the thumb and index finger Fig. Third, the cytobrush was removed while maintaining this grasp, and the soft tube and cytobrush were squeezed one time Fig.
Korea from four patients and a total of 24 samples 6 samples per patient. Separation of exfoliated cervico-vaginal cells from brush using elastic and soft tubes a insertion of the brush into the soft tube b squeezing of the tube and brush c removal of the brush.
We obtained five cervico-vaginal samples per patient and a total of 55 samples were analyzed. A soft elastic tube was used to separate exfoliated cells from the cervico-vaginal cytology samples as previous described methods.
We compared the RNA concentration and quality of each of the five types of samples for each patient using previously described methods. Primer sequences are described in Additional file 1 : Table S1. All PCR amplifications were performed with appropriate positive and negative controls.
The electrophoresis images of PCR products were analyzed and quantified using ImageJ, an open source image processing program designed for the analysis of scientific images. Statistical analysis was performed using SPSS version The median RNA concentration was significantly higher in soft tubes The mean concentrations of Group 1 Moreover, The sample letters a, b, c, d, and e indicate non-significant differences between groups.
The mean E6 densities of Group 1 37, This study provides a comparative analysis of the mRNA quantity and quality of cervico-vaginal cytology samples as affected by tube type, storage temperature, and preservative solution. Soft tubes enhanced the mRNA quantity and quality compared to the conventional rigid tubes usually used in cervico-vaginal cytology. We developed a novel method to separate exfoliated cells from cervico-vaginal smears from the brush using soft, elastic tubes.
We could separate the exfoliated cells from the brush by squeezing the soft tube. The quantity and quality of the mRNA extracted using the soft tube was excellent compared to that obtained from the conventional rigid tubes in this study. RNA samples obtained by cytology are partially degraded, leading to low levels of transcript detection [ 9 ]. RNases which enzymatically degrade RNA are nearly ubiquitous and pose a constant threat of contamination and degradation of purified RNA.
Values higher than this may indicate contamination with the aforementioned compounds. Despite the best efforts of the researcher, residual contaminants often remain in solution with nucleic acids following chemical isolation.
The presence of these can lead to an incorrectly high concentration reading or the disruption of downstream processes. DNA is a common contaminant of proteins isolated from whole cell lysates. Higher ratios may indicate the contamination of isolated proteins with DNA. Alternatively, the buffer used to isolate the sample protein may include components that absorb strongly in the UV region.
Note: Colorimetric spectrophotometric methods such as the BCA or Bradford assays are generally recommended when working with buffers with UV region absorbance.
If comparing ratios for samples measured on the microvolume mode and then diluted to be measured with a cuvette, ensure that the undiluted and diluted sample are at the same ionic strength and pH. It is not unusual to observe slight differences in purity ratios when measuring the same sample on different spectrophotometers. Up to about a 0. The absorbance of nucleic acid at nm is measured within a plateau region of the spectrum, while the nm absorbance is generally measured on a steep sloped portion of the spectral curve.
Suggested extraction methods for various organisms are listed on the Nanopore Sequencing Technology page. A gel image must be submitted along with all DNA samples. The gel must contain a size ladder, which should have an upper marker greater than or equal to 40 kbp e. The gel should be run slowly and sufficiently long so that it will be possible to determine if any sheared DNA exists in the sample.
A second measure of DNA purity that is often used for long read sequencing is a comparison between the sample concentration as determined by fluorometric e.
Qubit and UV-based e.
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