Background
Basic biological science research deals with nucleic acid isolation. Post-isolation nucleic acid integrity has a pivotal role in further elucidating gene expression and other molecular mechanisms. RNA (ribonucleic acid), cDNA (complementary deoxyribonucleic acid), and PCR (Polymerase chain reaction) products' integrity and quality are affected by several factors in biochemical and biophysical degradation modes. Inadequate evidence was noted about the direct effects of sodium hypochlorite and L-ascorbic acid. Objectives: This study aims to test the effects of sodium hypochlorite (SHC) and L-ascorbic acid (LAA) in total RNA and PCR products, respectively, in an acellular condition.
Conclusions
SHC (0.25%) is favorable in removing RNases and maintaining the integrity of RNA. cDNA synthesis did not affect by SHC treatment, and it follows the same as untreated samples after DNase 1 treatment. LAA drew a positive impact to improve the quality of PCR products in terms of band intensities, which is insignificant in SHC-treated RNA. Interestingly, it was revealed from our study that 5-25 µM LAA has the most beneficial role in the acquisition of PCR products, i.e. gene expression. These concentrations can be safely used to improve the quality of gene expression. This phenomenon can be used to achieve other, rarer, desired gene expressions. Further research is needed to explore the effects of SHC on the acquisition of PCR products using other solutions.
Methods
The study was categorized into three steps total RNA, cDNA, and PCR product evaluations. mBM-MSCs were used to extract RNA and then treated with SHC. Crude total RNA and, after DNase 1 treatment, the bands of total RNA samples were visualized by agarose gel electrophoresis. cDNAs were synthesized from SHC-treated (0.25%) and untreated RNAs, which were also expressed on the gel. LAA (5 µM, 15 µM, 25 µM, and 50 µM) were added to cDNAs synthesized from SHC- and non-SHC-treated samples. Housekeeping genes, Gapdh (Glyceraldehyde 3-phosphate dehydrogenase), and 18S rRNA (18S Ribosomal ribonucleic acid) were amplified in both groups.
Results
SHC-treated samples produced clearer bands on an agarose gel. Its treatment did not affect the integrated densities of agarose bands which revealed non-significant (P ≤ 0.05) differences in SHC-treated, untreated RNA, and cDNA. However, significant variations were observed at the PCR level. SHC-treated samples expressed decreased housekeeping gene expression in amplified products (Gapdh and 18S rRNA) and slightly but non-significantly high band intensities appeared in the presence of LAA. Significant variable differences (*P ≤ 0.05) were observed between SHC-treated and non-treated groups after LAA treatment. Conclusions: SHC (0.25%) is favorable in removing RNases and maintaining the integrity of RNA. cDNA synthesis did not affect by SHC treatment, and it follows the same as untreated samples after DNase 1 treatment. LAA drew a positive impact to improve the quality of PCR products in terms of band intensities, which is insignificant in SHC-treated RNA. Interestingly, it was revealed from our study that 5-25 µM LAA has the most beneficial role in the acquisition of PCR products, i.e. gene expression. These concentrations can be safely used to improve the quality of gene expression. This phenomenon can be used to achieve other, rarer, desired gene expressions. Further research is needed to explore the effects of SHC on the acquisition of PCR products using other solutions.