RNA template is one of the necessary raw materials for reverse transcription. At the same time, RNA templates under different conditions will also have a great impact on the reverse transcription process. The following are several factors that affect the quality of RNA templates.
RNA template self structure (high GC, complex secondary structure)
Due to the folding of its own sequence, RNA has different and complex secondary structures: paired double-stranded RNAs are helix, hairpin loop, protruding loop, inner loop, multi-branched loop and other structures. Because the paired bases are different, the stability of their pairing is also different. For example, the pairing between G and C, three pairs of hydrogen bonds, are the most stable. A and U are two pairs of hydrogen bond interactions; G and U are a pair of hydrogen bond interactions, which are the most unstable. Therefore, for RNA templates, the higher the GC content, the more complex the secondary structure.
In the process of reverse transcription, when the primer extends to the place with a secondary structure, the reaction will be forced to terminate, affecting the length of cDNA synthesis. At this time, it is recommended to incubate the template at 65 °C for 5 min and then rapidly cool on ice to denature the secondary structure; at the same time, the formation of RNA secondary structure can be reduced by reacting at a higher temperature (such as 55 °C).
RNA template purity and concentration
RNA purity will greatly affect reverse transcription experiments, such as salts, metal ions, ethanol, phenol, etc. mixed in the RNA purification process are common reverse transcriptase inhibitors. In addition, polysaccharides, polyphenols and humic acids in plant tissues can also inhibit reverse transcriptase.
For the determination of RNA purity, Nanodrop is usually used for determination. RNA with intact purity: 1.8 < OD260/OD280 < 2.0 (<1.8 indicates protein or phenol contamination, and phenol extraction can be increased; > 2.0 indicates that there may be residual is sulphuric acid); OD260/OD230 should be greater than 2. (<2 indicates residual guanidine isothiolate, β-mercaptoethanol or ethanol; reprecipitation can be performed, and ethanol washing can be repeated).
For the determination of RNA concentration, Nanodrop is usually used for determination.
RNA template integrity
The integrity of RNA greatly affects the length and quality of cDNA synthesis. Special protective measures need to be taken during RNA extraction, storage, and experimentation, such as operators wearing gloves and masks, and using RNase-free experimental consumables throughout the process.
For downstream cloning experiments, RNA integrity will affect the length of cDNA synthesis, thereby affecting the synthesis of the target gene. For downstream target gene quantification experiments, its CT value will be seriously affected, thereby affecting the accuracy of quantitative results (see the figure below).
The relationship between RNA RIN value and CT value
The integrity of RNA is usually detected by agarose gel electrophoresis. For the ratio of 28S and 18S rRNA to evaluate the integrity of RNA, close to 2:1 is relatively complete RNA.
Removal of gDNA from the RNA template
During the downstream qPCR experiment, the gDNA mixed in the Total RNA can be directly used as the template of the PCR reaction for amplification, resulting in false positives in the experimental results. Therefore, the RNA template must be de-gDNA processed before reverse transcription to ensure that there is only cDNA in the template.
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