Reverse Transcription Solutions

As the saying goes, "well begun is half done", but we also have a saying that "everything is difficult at the beginning", and this saying is fully reflected in RT-PCR. The first step - how to efficiently reverse RNA into cDNA, seems simple on the surface, but in fact it is full of traps. If you are not careful, it will make the high-spirited experimental masters fall into the trap without knowing how they fell into the trap. Obviously, it is not that simple to efficiently reverse transcribe RNA into cDNA. Today, I will share with you some tips in reverse transcription experiments.

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

• Do 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 to remove the secondary structure?

• How to choose primers for reverse transcription?

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

RNA concentration, purity, and integrity

In the previous soft article "Extraction and Quality Control of Nucleic Acids in qPCR Experiments", we gave a detailed introduction to the concentration, purity and integrity of RNA. You can review the content of our previous issue. The purity and integrity of RNA not only affect the efficiency of reverse transcription, but also determine the accuracy of quantitative experiments. First of all, salt, metal ions, ethanol and phenol residues in RNA samples will inhibit the efficiency of cDNA synthesis reactions. Therefore, it is very necessary to purify RNA in RNA samples containing impurities; secondly, the integrity of RNA is also a very important 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 sequences at the 3' end, and the 5' end or the middle fragments are not effectively reverse transcribed. If we set the position of the primer close to the 5' end or the middle, it is obvious that the result is much smaller than the actual one. Secondly, for full-length gene reverse transcription, complete 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 in reverse transcription can be from ng to several μg, and can be adjusted according to the actual situation of the experiment. Of course, the first principle to be followed 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 genes with low abundance expression, the starting amount of reverse transcribed RNA can be appropriately increased; finally, the RNA dosage range recommended by the kit used should be followed. Too low or too high 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 samples can be treated with DNase I to remove the residual DNA in the samples. Most of the finished reverse transcription kits on the market today contain components other than genomic DNA, so this problem is usually easy to solve.

Reverse transcription of GC-rich genes

Reverse transcription experiments of target genes with high GC content may be a headache for us. The reverse transcription efficiency of general enzymes for these high GC fragment genes is usually relatively low. Now there are some enzymes on the market that can withstand higher temperatures after genetic engineering modification, and have better reverse transcription efficiency for high GC fragments. Therefore, for such experiments, you can choose the corresponding kit; in addition, for reverse transcription experiments of these high GC genes, you can pre-treat them at 65℃ for 5 minutes, and then add reverse transcriptase to synthesize cDNA, which can improve the efficiency of reverse transcription.

RNA secondary structure

The secondary structure of RNA is one of the factors that hinder the effective synthesis of cDNA (Figure 2). For a specific RT-qPCR experiment, it is best to know whether the RNA of a gene we are testing contains a secondary structure, so as to determine our strategy in the reverse transcription experiment. Here we recommend a web-based RNA secondary structure prediction software - Mfold (Figure 3), which allows you to more intuitively understand the RNA situation 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 to reverse the reaction. This can improve the efficiency of RNA reverse transcription. The disadvantage is that you cannot get very long cDNA (as shown in Figure 4), but it does not have a big impact on qPCR.

Figure 4: Mixing Oligo dT and Random Primer to Improve Reverse Transcription Efficiency

Second, reverse transcription is performed under high temperature conditions (Figure 5). The advantage of using high temperature to open the secondary structure is that the reverse transcription efficiency of the secondary structure RNA can be improved, and a longer cDNA can be obtained at the same time. The disadvantage is that the thermal stability of the enzyme is very high, which will reduce the reverse transcription efficiency of the enzyme. General enzymes are easily inactivated at high temperatures. In addition, some secondary structures cannot be completely opened under such conditions.

Figure 5. Reverse transcription under high temperature conditions

3. Two-step method for reverse transcription (as shown in Figure 6): first incubate at high temperature to open the RNA secondary structure, then add reverse transcriptase for reverse transcription reaction. The advantage of this method is that it can completely open the RNA secondary structure and improve the reverse transcription efficiency, and it does not affect the activity of reverse transcriptase. The disadvantage is that the operation is slightly more complicated.

Figure 6. Schematic diagram of two-step reverse transcription

Primer selection

Random primers are mostly used for reverse transcription of RNA in prokaryotes, and are also used in eukaryotes, especially in reverse transcription experiments of RNA in complex templates or degraded templates. However, it can synthesize cDNA using rRNA as a template, which has the risk of overestimating the copy number.

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

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

Gene-specific primers have the best specificity for cDNA synthesis and are the first choice for qPCR.

Pay attention to details in experimental operation

RNA will be degraded by RNaseA, which is ubiquitous, so when extracting RNA and reverse transcribing, ribonuclease contamination must be avoided.

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°C.