How to verify the quality of RNA

qPCR evaluates gene activity by measuring the transcription level of RNA.The quality of RNA sample extraction directly affects gene expression analysis.There are three factors that affect RNA quality: RNA concentration, RNA purity and RNA integrity.

The concentration, purity, and integrity of RNA can be measured in several ways:

Spectrophotometer:

RNA concentration was calculated by measuring the 260nm absorption value, and the ratio of 260nm/280nm absorption value was measured to evaluate RNA purity.It is important to note that nucleic acid substances should be tested in a fixed pH solution.

The 260nm/280nm ratio ranges from 1.9 to 2.1.< 1.9, indicating protein residue;> 2.1, indicating possible RNA degradation.

Agarose gel electrophoresis:

Complete RNA usually has three bands, the brightest being the 28S band, followed by the 18S band, and the lightest being the 5S band (the 5S band is filtered out when some kits are extracted).The ratio of 28S to 18S can be measured by agarose gel electrophoresis.This method is mainly used to detect the purity and integrity of RNA.

If the 28S and 18S bands are bright, clear, and sharp, and the brightness of 28S is more than twice that of the 18S bands, we consider RNA to be of the best quality.

If the RNA bands appear diffuse, the reasons may be: RNA is degraded by nuclease, too high voltage or current, too high or too low sample loading.

The above two methods are commonly used in the laboratory, and there are several methods for you to choose from:

Fluorescent dye detection:

The fluorescence activity was enhanced by the combination of fluorescent dye and RNA.This method has high sensitivity and is mainly used to detect the concentration of RNA.

Microcapillary electrophoresis:

The RIN (RNA Integrity Number) score that can be evaluated is 10 for best RNA Integrity and 0 for worst RNA Integrity.This method has high sensitivity and resolution, and can be used to detect the concentration, purity and integrity of RNA.

3 '-5' integrity test:

GAPDH mRNA integrity is detected in a RNA sample to represent the integrity of all RNA. It is mainly used to test RNA integrity.

Tips for getting high-quality RNA:

1、Try to avoid contamination by RNA enzymes, use consumables and reagents without RNA enzymes, and it is recommended to separate the sites before and after amplification.

2、minimize the sampling time, the sample is quickly frozen after treatment, and RNA stabilization solution can be added.

3、RNAse inhibitors can be used during RNA extraction.

4、Avoid freeze-thaw cycles.

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