As its name indicates, it is a derivative of formamide, the amide of formic acid. DMF is a polar hydrophilic aprotic solvent with a high boiling point. Formamide is a reagent that is an ionizing solvent in aqueous buffers. Procedures have been reported for the use of formamide in DNA sequencing and in polyacrylamide sequencing gels, which helps to eliminate secondary structure in nucleic acids and thus compressions in the gel data. The formamide is known for its ability to lower the T m of DNA [30], thus the DNA denatures in the lower temperature than the melting temperature.
Water soluble. Sensitive to light. Propanol, butanol, formic acid, formamide are polar solvents. Formamide is incompatible with strong oxidizers, acids and bases. Reacts with water very slowly at room temperature, but rate is accelerated by acids and bases at elevated temperatures. Why Formaldehyde is used in RNA gel? Category: science biological sciences. Formaldehyde serves primarily as a denaturing agent for RNA during agarose gel electophoresis.
An additional useful property of formaldehyde is its inhibitory effect on RNases [5], which helps maintain RNA integrity during separation and gel handling.
How do you measure RNA quality? How do you make RNA gel? Is RNA negatively charged? For quick analysis of RNA integrity, our lab has often replaced formaldehyde gel electrophoresis with the use of standard TAE-based agarose gels normally used in analysis of DNA. It is unclear whether the degraded RNA in these gels was due to poor quality RNA preparation or because RNases were present on the apparatus and degraded the sample while the gel was running.
It is the aim of this paper to introduce an alternative approach for analyzing RNA quality by gel electrophoresis.
Although it is unlikely that this degree of contamination would occur naturally, the use of this large amount of RNase A demonstrates the strength of bleach towards inactivating RNases. The agarose gels were then heated to melt the agarose and cooled. The presence of 28S, 18S, and 5. Increasing rRNA band integrity occurs with bleach concentrations of 0. The 28S, 18S, and 5. An increase in bleach concentration also results in a linear increase in amperage.
With the addition of bleach at concentrations of 0. When assessing the quality of RNA by gel electrophoresis, the presence of three distinct bands suggests high quality RNA. Transfer RNAs nt may or may not be visible [ 25 - 28 ]. With increasing bleach concentrations, the electrical resistance through the gel was decreased, resulting in a linear increase in milliamps mA.
At very high concentrations of bleach, increased amperage was needed to reach the desired voltage Fig. Whether other brands of bleach may be effective remains unknown. Moreover, if the bleach were added after the agarose was melted, the bleach could interfere with the setting of the gel and have deleterious effects on the ethidium bromide used to stain the RNA data not shown.
By adding the bleach prior to melting the agarose suspension, the aforementioned conflicts were circumvented. For these reasons, our short protocol Table 2 is an economical and convenient option for analyzing RNA integrity. While using readily available and cost-effective reagents, this new procedure minimizes preparation time, is simple to execute, and reduces the amount of toxic agents used.
The authors declare no competing interests. National Center for Biotechnology Information , U. Author manuscript; available in PMC Jul 2. Patrick S. Aranda , Dollie M. LaJoie , and Cheryl L. Author information Copyright and License information Disclaimer. Cheryl L. Copyright notice. See other articles in PMC that cite the published article.
This resulted in comparable separation of RNA ladders Fig. The TT buffer particularly improved the separation of large pre-RNAs by enhancing the relative mobility of bands in the top area of the gel Fig 1D , right panel. We used a 7. Lanes 3—5, total RNA extracted from Saccharomyces cerevisiae cells.
Lanes 6—7, total RNA from mouse 3T3 fibroblasts. Equal amounts of RNAs were loaded on all gels. Positions of 45S An increased separation of high molecular size fragments of double-stranded DNA was not noted during electrophoresis in p K -matched buffers [ 4 ]. The improvement in resolution observed in electrophoresis of RNA may therefore reflect a better ability of the HT and TT buffers to control migration of RNA molecules during separation under denaturing conditions.
For instance, components of the buffers might interact with RNA and play a role in maintaining RNA molecules in a denatured state. A higher buffering capacity [ 4 ] and balanced ionic composition of these buffers may also help to prevent ion exhaustion in gel areas where large RNA molecules migrate, thereby contributing to their separation according to molecular size. Furthermore, unlike photosensitive MOPS solutions [ 2 ], these buffers are stable under ambient light conditions.
Because the HT and TT buffers allowed rapid electrophoretic separation of long RNAs, we next tested whether we could reduce the amount of formaldehyde used in the procedure. Formaldehyde serves primarily as a denaturing agent for RNA during agarose gel electophoresis. An additional useful property of formaldehyde is its inhibitory effect on RNases [ 5 ], which helps maintain RNA integrity during separation and gel handling. High concentrations 2. Others suggested that the reason for the high concentrations was to counteract the diffusion of formaldehyde out of gels during long runs [ 2 , 3 ].
However, it is important to tightly cover the gel casting assembly with plastic wrap during agarose solidification and only submerge gels in running buffer immediately before samples are loaded to prevent formaldehyde losses from the gels. The five-fold reduction in formaldehyde concentration in our system Table 1 helps reduce exposure to this toxic chemical. However, we observed an abnormal migration of some RNA samples during electrophoresis when EDTA was omitted from the loading dye, presumably due to the presence of residual metal ions interfering with RNA denaturation.
The following procedure reduces the number of pipetting steps and time required for sample preparation as compared with previously described procedures [ 2 , 3 ].
Filter through a sterile 0. When ready to proceed with electrophoresis, pipet the required amounts of RNA dissolved in formamide into microtubes or PCR strips. Loading dye mixed with formaldehyde is not stable upon storage and must be used within a few hours. In conclusion, the method described here provides an easier way to run agarose-formaldehyde gels and helps to resolve long RNAs. The entire procedure requires fewer reagents and manipulations compared with traditional protocols, works for different gel formats and, in our experience, is sufficiently robust to work even in novices' hands.
The HT buffer makes a good all-around conductive medium for the separation of a wide range of RNA species.
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