In the detection of RNA samples, it is necessary to ensure that RNase is removed from the cuvette, especially if such RNA is still to be recovered after spectrophotometry. This condition can be achieved by sequential washing with 0.1 M NaOH, 1 mM EDTA and RNase free water. A blank control was set for the spectrophotometer using a buffer of diluted RNA.

In a spectrophotometer using a UV-transparent plastic cuvette, the absorbance at 260 nm (A260) was measured to determine the concentration of RNA. To ensure meaningful results, the reading should be between 0.15 and 1.0. At 260 nm, the absorbance of one unit corresponds to a concentration of 44 μg per ml of RNA (A260 = 1 → 44 μg/ml; based on the standard 1 cm metering distance). This correlation is only valid for detection under neutral pH conditions. Therefore, if you want to dilute the RNA sample, you should use a neutral pH low salt buffer (eg 10 mM Tris•Cl, pH 7.0).

In the detection of RNA samples, it is necessary to ensure that RNase is removed from the cuvette, especially if such RNA is still to be recovered after spectrophotometry. This can be achieved by sequential washing with 0.1 M NaOH, 1 mM EDTA and RNase free water. A blank control was set for the spectrophotometer using a buffer of diluted RNA. An example of a calculation in the quantification of RNA is as follows:

RNA quantification example
Volume of RNA sample = 100 μl
Dilution = 10 μl RNA sample + 490 μl 10 mM Tris•Cl, pH 7.0 (1/50 dilution ratio)
Measure the absorbance of the diluted sample using a 1 ml (RNase-removed) cuvette
A260 = 0.2

RNA concentration
= 44 μg/ml x A260 x dilution factor
= 44 μg/ml x 0.2 x 50
= 440 μg/ml

Total RNA
= concentration x sample volume in milliliters
= 440 μg/ml x 0.1 ml
= 44 μg RNA

RNA purity

The ratio between the 260 nm and 280 nm readings (A260/A280) reflects the purity of the RNA because contaminants such as proteins absorb UV light. However, the ratio of A260/A280 is also significantly affected by the pH. When using water without buffering capacity, the ratio of pH to A260/A280 results in a wide range of changes. A lower pH will result in a smaller A260/A280 ratio and a lower sensitivity to protein contamination (3).

In order to obtain an accurate ratio, we recommend measuring the absorbance in a low base buffer of microalkali (eg 10 mM Tris•Cl, pH 7.5). In the 10 mM Tris•Cl, pH 7.5 buffer, the A260/A280 ratio of pure RNA was approximately 1.9–2.1.

Note: Some spectrophotometers can routinely achieve a ratio of up to 2.3 when detecting pure RNA.

Be sure to calibrate the spectrophotometer with the same solution. However, in order to accurately determine the concentration of RNA, we still recommend using a neutral buffer to dilute RNA samples, since the relationship between absorbance and concentration (A260 reading 1 corresponds to an RNA concentration of 44 μg/ml) is based on neutrality. The absorbance coefficient obtained by the condition.

RNA integrity

The integrity and size distribution of total RNA can be detected by denaturing agarose gel electrophoresis, ethidium bromide staining or a commercially available detection system such as the QIAxcel system or Agilent 2100. The enriched region of each ribosomal RNA should appear as a sharp band after gel staining. The band brightness of 28S ribosomal RNA should be about twice that of the 18S rRNA band. If the ribosomal RNA band in a lane is not sharp enough, but the dispersion of small-sized RNA occurs, it is likely that the RNA sample has undergone severe degradation during the preparation process.

The Agilent 2100 Bioanalyzer also provides the RNA Integrity Number (RIN) parameter as a useful measure of RNA integrity. Ideally, the RIN value should be close to 10, but in many cases (especially for tissue samples), RNA quality depends primarily on the preservation of the original sample.

Ribosomal RNA size from different sources

Principle of denaturing gel analysis

RNA can be isolated and identified using the charge transport effect of RNA in a formaldehyde agarose gel. Unlike DNA, RNA has a complex secondary structure and therefore requires the use of denaturing gels. Formaldehyde in the gel is able to disrupt the secondary structure of the RNA, allowing the RNA molecules to be efficiently separated in a manner consistent with charge transport.

In an electric field, the negative charge carried on the backbone phosphate group causes the nucleic acid molecule to move toward the anode. The rate of migration of denatured RNA molecules depends on their size; however, the fragment size is not linearly related to the rate of migration, as larger fragments encounter greater frictional resistance and movement in the gel is more difficult.

Agarose gel analysis is the most commonly used method of RNA analysis. Usually, the agarose gel with the appropriate resolution range is selected according to the size of the RNA. Small RNA fragments, such as tRNA or 5S rRNA, can be analyzed using polyacrylamide gel electrophoresis. The Molecular Biology Handbook (2, 4) can be consulted for detailed information on various types of analytical gels. This section will introduce formaldehyde agarose gel electrophoresis.

Preparation of formaldehyde agarose gel for RNA analysis

The following formaldehyde agarose (FA) gel electrophoresis protocol can increase the sensitivity of gel separation and subsequent detection (eg, Northern blotting). A key feature of this protocol is the use of a concentrated RNA loading buffer, which allows a larger amount of RNA sample to be loaded into the gel (compared to traditional protocols).

Agarose

The agarose used to prepare the gel is determined by the size of the isolated RNA fragment. For most RNA species of interest, the best results are obtained using a 1.0–1.2% (mass to volume) agarose concentration. For larger mRNA types, lowering the agarose concentration may be helpful. To separate smaller mRNAs, the agarose concentration can be increased to 2%. For smaller RNA species, such as tRNA or rRNA, polyacrylamide gel electrophoresis is recommended.

Use ultrapure agarose because impurities such as polysaccharides, salts, and proteins can affect RNA migration.

Tip: Some plastics in the electrophoresis tank are not resistant to ethanol. Please pay attention to this and check the manufacturer's instructions.

Total RNA-Formaldehyde Agarose Gel

10ug RNA per lane

The above information is from QIAGEN: http://