Reverse Transcription Solutions

As the saying goes, "Well begun is half done," but there's also a saying that "everything is difficult at the beginning," and this is particularly true in RT-PCR. The first step—efficiently converting RNA to cDNA—may seem simple on the surface, but it's actually fraught with pitfalls. A single mistake can leave even the most ambitious experimenters feeling bruised and battered, and they won't even know how. Clearly, efficiently reverse transcribing RNA to cDNA is no simple task. Today, we'll share some tips for reverse transcription experiments.

First of all, before reverse transcription, we need to think about some issues:

• Does the concentration, purity, and integrity of the RNA in the sample meet the requirements for reverse transcription?

• How much RNA should be added for reverse transcription?

• How to solve the problem of genomic DNA contamination?

• What is the GC content of the target gene we are quantifying? How can we effectively amplify genes with high GC content?

• Does the RNA of the target gene we want to reverse transcribe contain secondary structure? If so, how can we remove it?

• How to choose primers for reverse transcription?

• What details should we pay attention to during the operation?

RNA concentration, purity, and integrity

In our previous soft article, "Nucleic Acid Extraction and Quality Control in qPCR Experiments," we provided a detailed introduction to RNA concentration, purity, and integrity. You can review our previous article. RNA purity and integrity not only affect the efficiency of reverse transcription but also determine the accuracy of quantitative experiments. First, residual salts, metal ions, ethanol, and phenol in RNA samples can inhibit the efficiency of cDNA synthesis reactions. Therefore, RNA purification is essential for RNA samples containing impurities. Second, RNA integrity is also a crucial factor that determines our reverse transcription strategy. For example, for organisms, we usually use Oligo dT for reverse transcription experiments, which is usually feasible for RNA with good integrity (RIN > 7). However, for degraded RNA, the reverse transcription results are all 3' end sequences, and the 5' end or middle fragments are not effectively reverse transcribed. If we set the position of the primer close to the 5' end or the middle position, it is obvious that the result obtained is much smaller than the actual one. Secondly, for full-length gene reverse transcription, intact RNA is necessary. Finally, in the same experiment, the integrity of RNA in different groups needs to be maintained at the same level to ensure accurate quantitative results.

Starting amount of RNA reverse

The starting amount of RNA for reverse transcription can range from ng to several μg and can be adjusted based on the actual experimental conditions. Of course, the first principle to follow is the same starting amount principle: the same amount of RNA should be used for reverse transcription between different samples to reduce system errors. In addition, for detecting low-abundance genes, the starting amount of reverse transcribed RNA can be appropriately increased. Finally, the recommended RNA amount range of the kit used should be followed. Too low or too high an amount will affect the efficiency of reverse transcription (Figure 1).

Figure 1. Effect of the amount of starting RNA template on reverse transcription efficiency

Genomic DNA contamination issues

For samples contaminated with genomic DNA, the sample can be treated with DNase I to remove residual DNA. Most commercially available reverse transcription kits contain components that remove genomic DNA, so this problem is usually easily resolved.

Reverse transcription of GC-rich genes

Reverse transcription experiments for target genes with high GC content can be a headache. Conventional enzymes typically have low reverse transcription efficiency for these GC-rich genes. However, some commercially available enzymes have been engineered to tolerate higher temperatures and exhibit improved reverse transcription efficiency for these GC-rich genes. Therefore, for such experiments, appropriate kits can be selected. Furthermore, for reverse transcription experiments involving these GC-rich genes, pre-treatment at 65°C for 5 minutes before adding reverse transcriptase for cDNA synthesis can improve reverse transcription efficiency.

RNA secondary structure

RNA secondary structure is one of the factors that hinder efficient cDNA synthesis (Figure 2). For a specific RT-qPCR experiment, it is best to know whether the RNA of the gene we are testing contains secondary structure, which can then determine our reverse transcription strategy. Here, we recommend the web-based RNA secondary structure prediction software - Mfold (Figure 3), which provides a more intuitive understanding of the RNA structure of the gene we are testing.

Figure 2: Secondary structures in complex templates hinder synthesis

Figure 3. RNA secondary structure prediction software

For RNA templates containing secondary structures, we usually have three strategies to improve reverse transcription efficiency.

1. Use Oligo dT and random primers for reverse transcription. This can improve the efficiency of RNA reverse transcription. The disadvantage is that you cannot obtain very long cDNA (as shown in Figure 4), but it does not have a significant impact on qPCR.

Figure 4: Mixing Oligo dT and Random Primer improves reverse transcription efficiency.

Second, reverse transcription at high temperatures (Figure 5) utilizes high temperatures to open up secondary structures. This has the advantage of increasing the efficiency of reverse transcription of RNA with secondary structures and producing longer cDNAs. However, this approach requires high enzyme thermal stability, which can reduce the enzyme's reverse transcription efficiency. Typical enzymes are easily inactivated at high temperatures, and some secondary structures cannot be fully opened under these conditions.

Figure 5. Reverse transcription under high temperature conditions

Third, the two-step reverse transcription method (as shown in Figure 6) involves first incubating at high temperature to open up the RNA secondary structure, then adding reverse transcriptase to initiate the reverse transcription reaction. This method has the advantage of completely opening up the RNA secondary structure, improving reverse transcription efficiency, while also not affecting reverse transcriptase activity. The disadvantage is that the procedure is slightly more complicated.

Figure 6. Schematic diagram of two-step reverse transcription

Primer selection

Random primers are primarily used for reverse transcription of RNA in prokaryotes, but are also used in eukaryotes, particularly for reverse transcription of RNA from complex or degraded templates. However, they can synthesize cDNA from rRNA, which carries the risk of overestimating copy number.

Oligo dT is used in reverse transcription experiments in eukaryotes (RNA contains a Poly-A tail). Its specificity is better than random primers, but it is greatly affected by complex structures and degradation of templates.

Oligo dT and random primers are used in combination, which is less affected by complex structures and compensates for the low efficiency of Oligo dT synthesis of certain fragments far from Poly-A due to the length limitation of enzyme synthesis. They can usually be used in a 1:1 mixture.

Gene-specific primers provide the best specificity for cDNA synthesis and are the preferred choice for qPCR.

Pay attention to details in experimental operation

RNA will be degraded by RNaseA, which is ubiquitous, so ribonuclease contamination must be avoided during RNA extraction and reverse transcription.

General Tips for Avoiding RNase Contamination

• Because DEPC is a strong RNase inhibitor, all tubes and pipette tips used for cDNA synthesis should be treated with DEPC or certified nuclease-free laboratory equipment should be used during RNA extraction and reverse transcription experiments.
• Wear gloves when handling RNA and all reagents, as skin is a common source of RNases. Change gloves frequently.
• Use RNase-free reagents, including RNase-free ultrapure water.
• Use RNase Inhibitor (provided with the kit) to protect RNA from RNases.
• During the reverse transcription reaction, all tubes should be kept sealed; when not performing experiments, all reverse transcription reagents should be sealed and stored at -20℃.