cDNA stands for complementary DNA, and can be described as a double stranded DNA that is made with RNA as template. Our ultimate goal is the sequence the RNA that we have extracted - and with our sequencing method of choice, we need to turn the RNA back into DNA for it to be read properly. To do this, we use a technique known as reverse transcription to read and copy the RNA contents onto a newly made DNA strand, with no genetic information lost!

Experiment

Reverse transcription consists of 3 major steps - complementary DNA strand synthesis, RNA digestion and synthesis of second strand (IDT, 2018). The steps are described below:

Synthesis of complementary DNA strand:
Due to previous polyA addition to 3´OH, all RNA molecules have similar sequence at 3´ end which only differs in number of added adenine bases. This allows using polyT (VNP) primers (Oxford Nanopore) to anneal to RNA template and reverse transcriptase (SuperScript IV, ThermoFisher) can initiate the transcription.

A second, so-called strand switching primer is added to the reaction. This compensates for under-representations of 5´ends in cDNA by introducing an additional template and therefore protecting the terminal base pairs. Terminal transferase activity of the RT adds a number of deoxycytidine bases. The SSP primer is complementary to these bases and acts as an extended template for the RT, not only protecting the terminal bases, but also allowing to introduce sequence of choice into the newly synthesized first strand.

RNA template digestion:
RNA template needs to be removed before the second DNA strand can be synthesized. This is done by adding ribonucleases (RNAse Cocktail Enzyme Mix, ThermoFischer) into the reaction and incubating. The enzyme mix consists of RNase A and T1.

Second strand synthesis:
The second DNA strand is synthesized using LongAmp Taq Polymerase (NEB) incubated for one round. Primers used in the reaction are complementary to the sequences introduced by SSP and VNP primers.

Results

In order to call a synthesis successful, cDNA needs to be synthesized in sufficient quantity with no RNA contamination, as the RNA would interfere with subsequent sequencing.

Qubit measurement of DNA: Initially, our only criteria for cDNA synthesis was an amount of cDNA as measured by Qubit (Thermo Fisher). Typically, the cDNA amount would be equal to roughly twice the mass of the input RNA. For 125 ng of mRNA input we have synthesized (in an usual experiment) about 300 ng of cDNA.

Qubit measurement of RNA: Due to sequencing runs having rather low throughput and quite significant clogging of the pores, we decided to also measure RNA content of our cDNA samples. In most samples, significant amount of RNA were found, sometimes equal to the initial input of RNA. It was therefore neccessary to investigate the source of this error as decribed in Troubleshooting section.

Troubleshooting

Does mix of RNA/DNA interfere with Qubit measurement?

Hypothesis:

The measured RNA in the sample could be caused due to lack of specificity of Qubit dye eg. RNA dye actually has affinity to DNA and therefore shows RNA in our samples.

Experiment:

Samples containing only RNA or DNA in concentration of 10 ng/µl were prepared in triplicate and each measured with two different Qubit kits (RNA HS Kit, DNA HS Kit, Thermo Fisher).

Results:

The table below shows average measured values for each of the samples. Measuring RNA (or DNA) with corresponding Kit resulted in expected results eg. around 10 ng of RNA and very low amount of DNA. Similar results were seen for DNA sample. When DNA was added into RNA, the measured RNA amount decreased, which was the only measuring bias we have observed.

Sample	RNA [ng/µL]	DNA [ng/µL]	After adding DNA	After adding RNA
RNA sample 10 ng/µl	9.84	0.64	6.86	-
DNA sample 10 ng/µl	0.33	11.3	-	11.7

Table 1: The average of measured values for each of the samples. Measuring RNA (or DNA) with corresponding Kit resulted in expected results eg. around 10 ng of RNA and very low amount of DNA.

Conclusion

The experiment only investigated interactions between dsDNA and ssRNA, which are not supposed to introduce any bias into the measurement as per manufacturer's information (Source). The remaining question is how would a RNA:DNA hybrid be treated by the dye. It is possible that RNA was not properly digested and therefore remains in the hybrid form. A hybrid could hypothetically be detected by both RNA and DNA specific kit. Unfortunately no proven hybrid sample was available and therefore this hypothesis could not be tested.

It was concluded that having DNA - RNA mixture does not influence the measurement in a significant way. RNA amount is moderately decreased after addition of DNA into the sample, which would not explain the presence of RNA in cDNA samples. What remains to be investigated is how would a RNA:DNA hybrid influence the measurement. We therefore conclude that the RNA measurement in our samples is accurate and there is RNA present, either as a ssRNA of RNA:DNA hybrid.

Is RNA template properly digested? Is it carried over with the AMPure XP Beads?

Hypothesis:

RNA is found in the final cDNA product as it is not digested properly. The Rnase Cocktail contains RNAse A and T1, which are only supposed to introduce nicks into the RNA. Provided that LongAmp Taq polymerase does not degrade these fragment and that the purification beads (AMPure XP, Agencourt) do have affinity to both RNA and DNA, the RNA would be found in the sample.

Experiment:

This troubleshooting experiment had three major parts: i) optimizing the protocol for the use of RNase Cocktail using longer incubation time and/or larger quantity, ii) use of RNAse H for digestion and iii) clean-up step with purification beads to determine whether the beads have affinity for RNA.

1. Total of 8 samples with 125 ng of RNA were prepared (from previous cDNA samples that shown RNA contamination, eg. there was DNA present in the samples as well). The experiment was optimized for time and amount of RNase Cocktail.

2. RNA ladder was digested using both RNase H, RNAse Cocktail or both. Also each of the tests was prepared in either RNase-free water or buffer solution used during the cDNA synthesis. Digested DNA was visualised on a gel.

3. 1000 ng of RNA ladder were purified using AMX beads and shown on a gel.

Results:

Overall, it was seen that subsequent digestion of RNA contaminated samples was successful regardless of used concentration as shown below. Digestion of RNA ladder using RNase H and/or Rnase Cocktail shows complete digestion for RNase Cocktail and RNase H seems not to be working. The beads do carry over most of the RNA including rather small fragments (200 bp). The detailed results of each experiments are shown below:

1.

Sample No.	Incubation Time [min]	RNase Cocktail [µl]	RNA after treatment
1*	10	1	Not Detectable
2	10	2	Not Detectable
3	10	0,5	Not Detectable
4	30	1	Not Detectable
5	30	2	Not Detectable
6	30	0,5	Not Detectable
7	45	1	Not Detectable
8	45	2	Not Detectable
9	45	0,5	Not Detectable

Table 2: The table shows treatment for 9 samples of RNA and DNA mixture, each containing 125 ng of RNA. *Corresponds to experimental set-up used during the actual cDNA synthesis.

According to measurement with Qubit RNA HS, all samples regardless of treatment show all RNA being degraded including sample, where treatment is identical to the one used in the actual experiment.

2. The gel below shows results of digestion of RNA ladder with the available RNases, Rnase H (line 5 and 6) and RNase Cocktail (line 7 and 8) or both (line 8 and 9).

From this experiment we can conclude that the buffer has no effect on digestion since sample in water and in reaction buffer appear the same. It can further be said that RNase Cocktail efficiently degrades the ladder, which is composed of ssRNA. RNAseH seems to not digest the ladder at all.

This corresponds with described ability of RNAseH to preferentially digest RNA:DNA hybrids.

Figure 1: Results after digestion with RNases.

3. The gel showed very significant carry over of RNA during the purification with AMPure beads (image not shown), which corresponds to manufacturer's information. If RNA was not properly digested, it would therefore be carried together with the cDNA during the entire library prep process.

Conclusion

Overall, the results of this troubleshooting procedure present results conflicting with result of the actual experiment. Here the RNases are shown to work very efficiently. Most likely, RNA used in troubleshooting has different properties that the RNA template in the cDNA synthesis (could be in a form of hybrid or some other unusual form) which leads to different digestion results.

Discussion

During the cDNA synthesis it was possible to achieve sufficiently high yields, usually exceeding double of the input mRNA amount. This cDNA was used to prepare sequencing libraries. As was shown later, it unfortunately contained undigested RNA, which significantly decrease the quality of sequencing results. We have therefore spend significant amount of time searching for the source of RNA contamination as reported in this section.

The experiments above show very contradicting results. In section i) it can be seen that treatment of cDNA with RNAse Cocktail after synthesis resulted in complete clearance of RNA from the sample. RNase Cocktail has also been shown to digest RNA ladder as visualized on the gel in Figure 1. The same enzyme has always been used during the cDNA synthesis procedure and it remains unclear why does it efficiently digest RNA after synthesis or RNA ladder and would not work during the actual synthesis.

One hypothesis that we considered was the presence of RNA:DNA hybrids, which would decrease efficiency of RNAse Cocktail, which is more efficient in digesting ssDNA. RNAse H was therefore added to address this issue but clearance of RNA from samples did not significantly increase. Even more confusingly, digestion did work in some of the samples despite the content / treatment being identical.

Conclusion

We have managed to successfully synthesize complementary DNA to our mRNA samples, which unfortunately did contain undigested RNA. A protocol needs to be developed that assures all of the RNA has been removed from the sample prior to the preparation of the library. Moreover, additional troubleshooting needs to be performed to determine why is the digestion not efficient.

References

[1] IDT, Use of template switching oligos (TS oligos, TSOs) for efficient cDNA library construction, [online], 2018 https://eu.idtdna.com/pages/education/decoded/article/use-of-template-switching-oligos-(ts-oligos-tsos)-for-efficient-cdna-library-construction

[2] Write reference here