cDNA Conversion

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. 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.

Result

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.

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.

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.

Conclusion

Discussion: 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.

Conclusion: 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.