In order to create a “viral” genome, there are many steps and different processes that the sample must go through first to be prepared for sequencing. Normally the sample is subjected to toxic cell culturing in order to grow enough “virus” to utilize the unpurified culture supernatant for sequencing. Sometimes the sequencing is done straight to the unpurified fluids from sick patients as was done in the case of “SARS-COV-2.” Whatever method is used, the sample must be broken down to extract the RNA needed for sequencing. However, there is a bit of a problem. It is said that there is normally not enough material left at the end of extracting the RNA to sequence a genome so it is a necessity to create more. In order to create the required material, PCR must be used for amplification. Unfortunately, it is claimed that PCR can only be used to amplify DNA and not RNA. Thus, the only way to make this work is by using a process known as Reverse Transcription Polymerase Chain Reaction (RT-PCR) to change the RNA into DNA. From RT-PCR, the RNA is converted to what is called complementary DNA or cDNA by use of an enzyme known as reverse transcriptase, hence the RT in RT-PCR. This is done in order to create what is called the sequencing library:
Sequencing library: what is it?
DNA and RNA sequencing libraries
“A sequencing library can be made by starting from genomic DNA or from RNA. The workflow for the preparation of a DNA sequencing library consists of three fundamental steps:
- Fragmentation and sizing of the nucleic acid (DNA or RNA) to obtain fragments of a predifined length
- Attachment of the adaptors (adapters) to the extremities of the fragments
- Library quantification
In any RNA sequencing library there’s an additional step: the RNA conversion in cDNA. The fragmentation step can be done before or after the cDNA synthesis.”
The sequencing library is essentially a pool of DNA fragments with adapters attached. Working with RNA, such as when sequencing a “virus,” requires the additional step of cDNA conversion before this library can be prepared. This conversion into cDNA is done for a few reasons:
- RNA is considered more unstable than DNA.
- PCR amplification only works on DNA.
- Most, if not all, sequencing protocols are designed for DNA.
So what exactly is cDNA and how does one go about creating it? The theory to explain cDNA relies heavily on the “virus” fraud as the only way for cDNA to exist is either through the use of reverse transcriptase, an enzyme created through the “natural” processes of “retroviruses” as they hijack host cells (i.e. fiction), or as synthetically engineered DNA in a lab using the synthetically created reverse transcriptase (i.e. fiction). In other words, the only way cDNA exists is through the use of a fictional enzyme from a fictional “virus” either created from cell culture in a lab or through synthetic means by RT-PCR:
cDNA vs Genomic DNA
“Initially, it was observed that gDNA was always read and transcribed into mRNA, which guided protein formation and then was disposed. The notion that information might always flow from DNA to RNA to protein was somewhat jokingly referred to as the Central Dogma of molecular biology. Calling it that challenged scientists to find exceptions to this rule.
Virologists eventually did find one such exception. Retroviruses were discovered to have mechanisms for “reverse transcription.” This means that they can take RNA chains and produce DNA chains from them. In this way, during reverse transcription, information flowed backwards from RNA back to DNA. DNA that arises from this process is called complementary DNA (cDNA). cDNA is either produced by some viruses or synthesized in laboratories.
cDNA can be described as gDNA without all the necessary noncoding regions, which is how it gets its name as complimentary DNA.”
“When scientists use viral enzymes to make cDNA from RNA isolated from the cells and tissues that they are studying, it does not contain introns due to being spliced out in mRNA. cDNA also does not contain any other gDNA that does not directly code for a protein (referred to as non coding DNA).”
“In order to isolate cDNA, first the RNA of an organism must be isolated. Then, using a reverse transcriptase enzyme, cDNA can be made. This is the process retroviruses use to incorporate into their host’s cells. Retroviruses, such as Simian Immunodeficiency Virus (SIV) and Avian Myeloblastosis Virus (AMV), use their cDNA to produce mRNA in the host, leading to the production of viral proteins. This is possible because retroviruses use RNA as their genomic material instead of DNA, and it is reverse transcribed into the cDNA, which then undergoes normal transcription and leads to the viral protein in the host.”
As can be seen, the RNA is put through the magical process of RT-PCR using the reverse transcriptase engineered from a “virus” and created in a lab. To put this in perspective, let’s break this process down a bit using the “SARS-COV-2” genome as an example. In order to create the genome for “SARS-COV-2,” the unpurified BALF of one patient was put through deep meta-transcriptomic sequencing. The RNA was extracted from this unpurified BALF and a library was constructed to search for “aetiological agents.” Out of 384,096 contigs assembled by Megahit9, they found that the longest (30,474 nucleotides (nt)) was closely related to a bat SARS-like “coronavirus.” They used RT-PCR to determine and confirm the genome and its terimini:
“Total RNA was extracted from 200 μl of BALF and a meta-transcriptomic library was constructed for pair-end (150-bp reads) sequencing using an Illumina MiniSeq as previously described4,6,7,8. In total, we generated 56,565,928 sequence reads that were de novo-assembled and screened for potential aetiological agents. Of the 384,096 contigs assembled by Megahit9, the longest (30,474 nucleotides (nt)) had a high abundance and was closely related to a bat SARS-like coronavirus (CoV) isolate—bat SL-CoVZC45 (GenBank accession number MG772933)—that had previously been sampled in China, with a nucleotide identity of 89.1% (Supplementary Tables 1, 2). The genome sequence of this virus, as well as its termini, were determined and confirmed by reverse-transcription PCR (RT–PCR)10 and 5′/3′ rapid amplification of cDNA ends (RACE), respectively. This virus strain was designated as WH-Human 1 coronavirus (WHCV) (and has also been referred to as ‘2019-nCoV’) and its whole genome sequence (29,903 nt) has been assigned GenBank accession number MN908947.”
Looking at the methods section, we find exactly what they used to convert the RNA into cDNA:
“Total RNA was extracted from the BALF sample using the RNeasy Plus Universal Mini kit (Qiagen) following the manufacturer’s instructions. The quantity and quality of the RNA solution was assessed using a Qbit machine and an Agilent 2100 Bioanalyzer (Agilent Technologies) before library construction and sequencing. An RNA library was then constructed using the SMARTer Stranded Total RNA-Seq kit v.2 (TaKaRa).”
It says that the library was created using the SMARTer Stranded Total RNA-Seq kit v.2 (TaKaRa). If we go to the manufacturer of this product, we can find this paper describing the library creation process which gives us the name of the RT that is used to create the cDNA:
“Random priming (represented as the green N6 Primer) allows the generation of cDNA from all RNA fragments in the sample, including rRNA. When the SMARTScribe Reverse Transcriptase (RT) reaches the 5′ end of the RNA fragment, the enzyme’s terminal transferase activity adds a few non-templated nucleotides to the 3′ end of the cDNA (shown as Xs).”
We find that they use a reverse transcriptase called SMARTScribe Reverse Transcriptase (RT). If we search for the product itself, we get this description detailing what it actually is:
“SMARTScribe Reverse Transcriptase is a high-performance enzyme that performs unbiased cDNA synthesis, allowing for amplification and library construction from any RNA transcript. SMARTScribe RT is a modified Moloney Murine Leukemia Virus reverse transcriptase that generates long, full-length cDNA (up to 14.7 kb) while preserving the relative transcript proportions of the original RNA sample. Our proprietary purification process and rigorous quality control standards ensure that virtually all contaminating nucleases have been removed from SMARTScribe RT. It has been specially formulated for use with all of our SMART kits.“
Thus, we can see that the RNA from the unpurified BALF was subjected to a modified Moloney Murine Leukemia “Virus”reverse transcriptase. While I couldn’t find the exact process used to create this specific RT, judging from the descriptions of numerous other M-MLV RT products out there, it was most likely generated synthetically in a lab by “isolation” from E. coli expressing a portion of the pol gene of the M-MLV on a plasmid gene. In other words, it is a recombinant genetically-modified creation from multiple sources. This RT was used to create the synthetic cDNA in order to sequence the genome of “SARS-COV-2.” We also see that the proprietary purification process can remove virtually all contaminating nucleases. In other words, it removes contamintion “in effect though not in fact; practically; nearly.” Otherwise stated as: it does not remove all contaminants. Adding reverse transcriptase generated from unpurified “virus” to create the cDNA is yet another impurity added to the sample before sequencing.
In order to believe in the legitimacy of the conversion of RNA to cDNA by way of RT-PCR, it must first be believed that murine leukemia “virus” and the avian sarcoma “virus” (as it was also used) were purified and isolated. As it is very clear no “virus” has ever been properly purified and isolated, this immediately torpedoes the legitimacy of this process. If they are unable to purify and isolate a “virus,” they would not be able to sequemce, identify, and isolate any genes said to belong to reverse transcriptase from said “virus.”
If we were to allow that they in fact isolated both MLV and ASV, we must then believe that these “viruses” were sequenced accurately and reliably without the use of sophisticated sequencing technology as RT was discovered in the early 1970’s which was well before the technological advancement of genomics with Sanger sequencing in the early 1980’s. Even today, sequencing technology said to be far more advanced is prone to limitations, errors, and unreproducible results. It stretches the imagination to believe that this was possible 50 years ago when it still isn’t possible today.
If we assume that the RT sequence was somehow correct, we must then believe that it was able to be synthetically recreated in a lab by combining the MLV pol gene with E. Coli. As detailed here, while this process was claimed to be done, it was not detailed in the original studies:
“The reverse transcriptase (RT) of Moloney murine leukemia virus (MMLV-RT) is the most widely used enzyme for cDNA synthesis and RNA amplification due to its robust catalytic activity and high fidelity (Kimmel and Berger 1987; Kievits et al. 1991). Although recombinant MMLV-RT has been produced in Escherichia coli (Tanese et al. 1985; Roth et al. 1985; Kotewicz et al. 1985), the expression and purification conditions were not detailed, and the four- to five-step purification strategy makes it necessary to design an alternative simple and quick method for producing this enzyme in a soluble and active form.”
The converting of RNA to cDNA takes many leaps in logic in order to believe that what is claimed to happen actually takes place. What occurs within these chemical reactions is entirely unobservable. The stories created around these experiments are entirely hypothetical. The legitimacy of this process hinges on the existence of an unseen “virus” used to create the synthetic reverse transcriptase enzyme in order to change RNA into synthetic cDNA so that a theoretical genome can be created for other unseen “viruses.” To say that belief in this process takes a great deal of blind faith is an understatement.
To gain a better understanding of this theoretical conversion, let’s take a look at the steps involved in the cDNA synthesis and see what we can find out. Presented below are the 5 steps Thermo Fisher provides to optimally create cDNA. Here you will be able to see the numerous alterations the RNA must go through as well as the chemicals added to the sample to generate the desired reaction:
5 Steps to Optimal cDNA Synthesis
“The synthesis of DNA from an RNA template, via reverse transcription, results in complementary DNA (cDNA). cDNA can then serve as template in a variety of downstream applications for RNA studies such as gene expression; therefore, cDNA synthesis is the first step for many protocols in molecular biology. If you are new to cDNA synthesis or experience researcher wanting to optimize your protocol, consider these five critical steps to help you ensure your cDNA synthesis results in highest efficiency.
Step 1. Prepare sample
RNA serves as the template in cDNA synthesis. Total RNA is routinely used in cDNA synthesis for downstream applications such as RT-(q)PCR, whereas specific types of RNAs (e.g., messenger RNA (mRNA) and small RNAs such as miRNA) may be enriched for certain applications like cDNA library construction and miRNA profiling.
Maintaining RNA integrity is critical and requires special precautions during extraction, processing, storage, and experimental use. Best practices to prevent degradation of RNA include wearing gloves, pipetting with aerosol-barrier tips, using nuclease-free labware and reagents, and decontamination of work areas.
To isolate and purify RNA, a variety of strategies are available depending on the type of source materials (e.g., blood, tissues, cells, plants) and goals of the experiments. The main goals of isolation workflows are to stabilize RNA molecules, to inhibit RNases, and to maximize yield with proper storage and extraction methods. Optimal purification methods remove endogenous compounds, like complex polysaccharides and humic acid from plant tissues that interfere with enzyme activity; and common inhibitors of reverse transcriptases, such as salts, metal ions, ethanol, and phenol. Once purified, RNA should be stored at –80°C with minimal freeze-thaw cycles.
Step 2. Remove genomic DNA
Trace amounts of genomic DNA (gDNA) may be co-purified with RNA. Contaminating gDNA can interfere with reverse transcription and may lead to false positives, higher background, or lower detection in sensitive applications such as RT-qPCR.
The traditional method of gDNA removal is the addition of DNase I to preparations of isolated RNA. DNase I must be removed prior to cDNA synthesis since any residual enzyme would degrade single-stranded DNA. Unfortunately, RNA loss or damage can occur during DNase I inactivation treatment.
As an alternative to DNase I, double-strand–specific DNases are available to eliminate contaminating gDNA without affecting RNA or single-stranded DNAs. Their thermolabile property allows simple inactivation at a relatively mild temperature (e.g., 55°C) without negative impacts. Such double-strand–specific, thermolabile DNases can be incubated with RNA for 2 min at 37°C prior to reverse transcription reactions to streamline the workflow (Figure 1 ).
Step 3. Select reverse transcriptase
Most reverse transcriptases used in molecular biology are derived from the pol gene of avian myeloblastosis virus (AMV) or Moloney murine leukemia virus (MMLV). The AMV reverse transcriptase was one of the first enzymes isolated for cDNA synthesis in the lab. The enzyme possesses strong RNase H activity that degrades RNA in RNA:cDNA hybrids, resulting in shorter cDNA fragments (<5 kb).
The MMLV reverse transcriptase became a popular alternative due to its monomeric structure, which allowed for simpler cloning and modifications to the recombinant enzyme. Although MMLV is less thermostable than AMV reverse transcriptase, MMLV reverse transcriptase is capable of synthesizing longer cDNA (<7 kb) at a higher efficiency, due to its lower RNase H activity.
To further improve cDNA synthesis, MMLV reverse transcriptase has been engineered for even lower RNase H activity (i.e., mutated RNase H domain, or RNaseH–), higher thermostability (up to 55°C), and enhanced processivity (65 times higher). These attributes result in increased cDNA length and yield, higher sensitivity, improved resistance to inhibitors, and faster reaction times (Table 1).
Step 4. Prepare reaction mix
In addition to enzyme and primers, the main reaction components for reverse transcription include RNA template (pre-treated to remove genomic DNA), buffer, dNTPs, DTT, RNase inhibitor, and RNase-free water (Figure 2).
Step 5. Perform cDNA synthesis
Reverse transcription reactions involve three main steps: primer annealing, DNA polymerization, and enzyme deactivation. The temperature and duration of these steps vary by primer choice, target RNA, and reverse transcriptase used.
The critical step is during DNA polymerization. In this step, reaction temperature and duration may vary according to the primer choice and reverse transcriptase used. If using random hexamers, then we recommend incubating the reverse transcription reaction at room temperature (~25 °C) for 10 min after enzyme addition to extend the primers.
Among reverse transcriptases there are differences in thermostability, which in turn determines the highest optimal polymerization temperature for each. Using a thermostable reverse transcriptase allows, a higher reaction temperature (e.g., 50°C), to help denature RNA with high GC content or secondary structures without impacting enzyme activity (Figure 3). With such enzymes, high-temperature incubation can result in an increase in cDNA yield, length, and representation.”
Now that we have the general outline of the steps involved with synthesizing cDNA from RNA, Thermofisher nicely highlighted some of the problems that can occur during this process:
Overcoming Hurdles in cDNA Synthesis
“You can tell a lot about a cell by looking at its RNA, but these transitory molecules are difficult to study directly. RNA degrades easily because the extra hydroxyl group in the ribose sugar is highly reactive. DNA is more stable because it has a deoxyribose sugar backbone and double-stranded structure. That’s why scientists use reverse transcription to make complementary DNA (cDNA), which captures the RNA sequences in DNA form. Making cDNA is the first step in many molecular biology applications—from cloning and RT-PCR to microarrays and next-generation sequencing.
To maintain the validity of experimental results, you need cDNA that faithfully represents the template RNA. Major hurdles in cDNA synthesis reactions include the secondary RNA structures that may slow or even halt reverse transcription. Degradation of template RNA by the intrinsic RNAase activity of reverse transcriptases (RTs) is another problem that leads to truncated cDNA. Also, inhibitors present in RNA samples may reduce polymerization activity of the reverse transcriptase enzymes. Template degradation and inefficient polymerization result in cDNA of low quality and quantity.
To boost enzyme performance, scientists at Thermo Fisher Scientific used molecular evolution and rational design to engineer a better reverse transcriptase. Starting with the wild-type RT gene from the Moloney murine leukemia virus (M-MuLV), our scientists introduced a number of improvements to help you achieve better cDNA yields, longer products, and greater representation of input RNA.
Enzyme boost #1: Increased thermostability
Single-stranded RNA forms hairpin loops and other secondary structures, which can interfere with cDNA synthesis. The ability of a reverse transcriptase to tolerate high temperatures can help overcome this challenge. Wild-type M-MuLV RT works at 37–42°C, while our modified enzyme maintains activity up to 50–55°C. Elevated reaction temperatures destabilize RNA secondary structures, allowing the RT to read through the sequence, thus increasing cDNA yield.
Enzyme boost #2: Diminished RNase H activity
The first strand of cDNA synthesis creates DNA-RNA hybrid molecules. RTs often have built-in RNase H activity—the ability to hydrolyze RNA before completing the second cDNA strand. However, too much RNase activity can degrade template RNA prematurely, which can lower the yield and length of cDNA products. Our RT includes modifications that alter or significantly reduce RNase H activity resulting in an increase yield of full-length cDNA synthesis products.
Enzyme boost #3: Higher processivity
Substances that bind to RNA and interfere with cDNA synthesis are commonly carried over from RNA sample sources. Processivity is a key enzyme feature that refers to the number of nucleotides incorporated during a single binding event. Increased processivity correlates with tighter substrate binding and can improve resistance to these inhibitors. High processivity lets RTs synthesize longer cDNA strands in a shorter reaction time. For our improved RT, it takes just 15–30 minutes for cDNA synthesis depending on whether genomic DNA is removed or not. This efficiency brings higher yield and superior sensitivity with low quality or low quantity RNA samples, even RNA from single cells.”
It can be seen from the above source that there are a few potential hiccups in the cDNA synthesis process such as:
- Secondary RNA structures which can halt or stop the RT process
- Degradation of template RNA by intrinsic RNase
- Reduced polymerization due to inhibitors
- Subtances carried over from sampling sources which interfere with cDNA synthesis
Even with the supposed “purification” of the RNA in the initial extraction steps for creating the genome, there are still problems associated with contamination throughout the remaining steps of this process. In RT-PCR, contamination is a persistent problem that leads to inaccurate data as PCR can not differentiate between synthesized cDNA and contamination. This problem can only be mitigated (i.e. made less severe) and remains without an easy remedy:
Avoiding DNA Contamination in RT-PCR
“A frequent cause of concern among investigators performing quantitative RT-PCR is inaccurate data due to DNA contamination in RNA preparations. Although DNA contamination is easily detected by performing a “no-RT” control, there is no easy remedy. In this technical bulletin, we present data showing levels of DNA contamination in RNA generated by different procedures, and suggest several precautionary measures that can be implemented to reduce the impact of this persistent problem.
RT-PCR and Genomic Contamination
RT-PCR is an increasingly popular method for the quantitative analysis of gene expression. With this popularity comes a heightened awareness that most techniques used for total RNA isolation yield RNA with significant amounts of genomic DNA contamination. PCR cannot discriminate between cDNA targets synthesized by reverse transcription and genomic DNA contamination.”
Another issue facing RT-PCR is that it is the main source of bias and artefacts during library construction. The number of cycles used can greatly influence the amount of bias generated as the higher the cycle, the greater the bias. It is considered essential to avoid bias yet this is rarely ever the case. These biases and artefacts make their way into the final genome leading to inaccurate, unreliable, and unreproducible results:
Bias in RNA-seq Library Preparation: Current Challenges and Solutions
3.6. Reverse Transcription
“Currently, the strategies of transcriptome analysis are still to convert RNA to cDNA before sequencing. A known feature of reverse transcriptases is that they tend to produce false second strand cDNA through DNA-dependent DNA polymerase. This may not be able to distinguish the sense and antisense transcript and create difficulties for the data analysis.”
3.7. PCR Amplification
“PCR is a basic tool widely in molecular biology laboratories. In particular, the combination of PCR and NGS sequencing promoted the explosive development of RNA sequence acquisition. However, PCR amplification has been proved to be the main source of artifacts and base composition bias in the process of library construction, which may lead to misleading or inaccurate conclusions in data analysis. Therefore, it is essential to avoid PCR bias, and great efforts have been expended on trying to control and mitigate bias in current.”
3.8. The Sources of PCR Amplification Biases and Improvement Methods
3.8.1. Extremely AT/GC-Rich
“Studies have been indicated that fragments of GC-neutral can be amplified more than GC-rich or AT-rich fragments. Therefore, the fragments with high AT or very high GC content often have little or no amplification at all [47, 48]. These unfavorable features result in difficulties in genome sequencing of extremely AT-rich, such as human malaria parasite , or high GC (Bordetella pertussis) genomes (average GC content, about 75%).”
3.8.2. PCR Cycle
“As we all know, PCR can exponentially amplify DNA/cDNA templates, thus leading to a significant increase of amplification bias with the number of PCR cycles . Therefore, it is recommended that PCR be performed using as few cycle numbers as possible to mitigation bias [52, 53]. At the present, several laboratories have compared different PCR cycle number to reduce amplification bias. Wu et al.  performed a comprehensive analysis. The results of the study indicate that comparing with the lower cycle number, the higher cycle number can produce significant biases or artifacts in standard amplifications of mixed templates. In addition, Sze and Schloss’s  study indicated that reducing the number of cycles of amplification can also decrease PCR biases and artifacts using a mock community and human stool samples.”
Finally, the reverse transcriptase is a source of contamination itself, which should not be all that shocking considering the source material used to create the recombinant concoction. In this 2011 study, five of the most used RT enzymes were investigated and it was observed that they all had non-specific cDNA amplification. Findings of RNA contaminants in the RT is hypothesized as the cause of this problem. This is an issue that is extensively described in virology and it easily leads to false-positive results as it can interfere with RT specificity:
Commercial reverse transcriptase as source of false-positive strand-specific RNA detection in human cells
“A commonly used technique to investigate the expression of an antisense ncRNAs is strand-specific reverse transcription coupled with polymerase chain reaction (RT-PCR). The advantage of this accurate technique is that it does not require any special equipment or expertise. The disadvantage is that it can lead easily to false-positive results. We applied strand-specific RT-PCR to investigate the presence of antisense ncRNA associated to Retinoic Acid Receptor Beta 2 (RARβ2) in different human tumoral cell lines. By performing this technique, we observed false-positive detection of ncRNA. For accurate interpretation of the results in RT-PCR experiments, we introduced a «No primer» control that reveals non-specific cDNA synthesis. Moreover, we report the presence of non-specific cDNA amplification with five of the most frequently used reverse transcriptase in absence of added primers. We found that the choice of the reverse transcriptase as well as the conditions of the reaction (RT temperature and PCR cycle number) are important parameters to choose as the different reverse transcriptases do not display the same cDNA synthesis background. This previously observed phenomenon was reported to originate from the «self-priming» of RNA template. Here, we report rather the presence of RNA contaminants associated with one of the reverse transcriptase studied that might contribute to non-specific cDNA synthesis.”
“The critical step of the procedure is the production of DNA from the RNA template by retroviral reverse transcriptase (RT) in the presence of sequence-specific or random oligodeoxynucleotide primers. To specifically identify antisense ncRNA overlapping sense transcripts but oppositely oriented, it is not possible to use random oligodeoxynucleotide primer RT-PCR since it cannot distinguish between the two species. To avoid this problem, strand-specific primers are used leading to the specific reverse transcription of the sense or the antisense RNA followed then by PCR amplification. For detection of. antisense RNA, only the primer complementary to this strand is added in the RT reaction in order to obtain cDNA synthesis only when the antisense transcript is present in the total RNA sample. Here we applied this technique to a chosen target and observed a non-specific amplification and false-positive results. These results were obtained with high reproducibility with five different commercial RTs on three genes in several human cell lines. This non-specific cDNA synthesis leading to strand-specificity aberrant detection was previously observed in the field of Virology where this approach was used to distinguish between negative and positive-strand viral RNA [13e16] and is known as «false-priming». Several mechanisms have been proposed to explain how cDNA synthesis comes from false-priming during the RT reaction, including random priming by contaminating endogenous or exogenous nucleic acids [14,17,18] and RNA secondary hairpin structures that can be recognized and extended by the RT enzymes, the so-called «self-priming» [17,19,20]. Here we demonstrate that such non-specific cDNA synthesis is a global phenomenon occurring also with human cellular RNA. Depending on the RT enzyme used, it leads to different levels of false-positive strand-specific RT-PCR results that can be misinterpreted in absence of appropriate controls. This is of fundamental importance in all applications looking for sense and antisense RNA strand-specific detection. In our experiments, we found small RNA contaminants in AMV RT commercial preparations that could be involved in falsely-primed cDNA synthesis.”
“During our investigation on the existence of RARb2 antisense ncRNAs, we came across an unexpectedly high level of false-posi-tive results. cDNA synthesis from cellular total RNA without adding primers revealed a global non-specific reverse transcription of the RNA template. This false-primed event has been well documented in the field of Virology, in which it is a high source of false-positive results during strand-specific viral RNA detection [13e16]. It is believed that the origin of primer-independent cDNA synthesis comes from either the presence of cellular nucleic acids contaminants like oligo(dT) , exogenous nucleic acid contaminants [14,17] or secondary structured RNAs [17,19,20].”
“Noteworthy, we showed that five of the most commonly used RT enzymes display non-specific cDNA synthesis, but the amount of background was not the same depending on the RT enzyme (Fig. 3). Therefore, this observation raised the possibility that non-specific cDNA synthesis came from the enzyme themselves rather than from the RNA template. We showed that reverse transcriptase RNase H activity was not implicated since cDNA synthesis was observed independently of an associated RNase H activity (Fig. 3).”
“As extensively described in virus, we showed that initiation of cDNA synthesis during RT-PCR can occur on human cellular mRNA without addition of any exogenous primers in the RT step. This non-specific cDNA synthesis is a global phenomenon, detected at different level with five of the most used commercial reverse
transcriptases. This can interfere with RT specificity and is a high source of false-positive results, especially in the discrimination of antisense from sense RNA.”
- The notion that information might always flow from DNA to RNA to protein was somewhat jokingly referred to as the Central Dogma of molecular biology
- However, this central dogma challenged scientists to find exceptions to this rule
- Virologists found the exception when “retroviruses” were discovered to have mechanisms for “reverse transcription”
- This means that they can take RNA chains and produce DNA chains from them and thus information flowed backwards from RNA back to DNA (how convenient…)
- DNA that arises from this process is called complementary DNA (cDNA)
- cDNA is either produced by some “viruses” or synthesized in laboratories
- cDNA can be described as gDNA without all the necessary noncoding regions
- Scientists use “viral” enzymes to make cDNA from RNA isolated from the cells and tissues that they are studying
- In order to isolate cDNA, first the RNA of an organism must be isolated (which is never done with “viruses”)
- Then, using a reverse transcriptase enzyme (an enzyme supposedly isolated from a “virus” never isolated) cDNA can be made
- This is the process “retroviruses” use to incorporate into their host’s cells (in other words, the existence of cDNA relies on unobserved hypothetical processes of non-existent “viruses”)
- This is possible because “retroviruses” use RNA as their genomic material instead of DNA, and it is reverse transcribed into the cDNA, which then undergoes normal transcription and leads to the “viral” protein in the host
- For example, the genome sequence of “SARS-COV-2,” as well as its termini, were determined and confirmed by reverse-transcription PCR (RT–PCR)10 and 5′/3′ rapid amplification of cDNA ends (RACE)
- An RNA library was then constructed using the SMARTer Stranded Total RNA-Seq kit v.2 (TaKaRa)
- They used SMARTScribe RT, which is a modified Moloney Murine Leukemia “Virus” reverse transcriptase
- The proprietary purification process and rigorous quality control standards ensure that virtually all (i.e. not all) contaminating nucleases have been removed
- In other words, the RT used to convert the “SARS-COV-2” RNA into cDNA was generated synthetically in a lab by “isolation” from E. coli expressing a portion of the pol gene of the murine leukemia “virus” on a plasmid gene
- Although recombinant MMLV-RT has been produced in Escherichia coli (Tanese et al. 1985; Roth et al. 1985; Kotewicz et al. 1985), the expression and purification conditions were not detailed
- A sequencing library can be made by starting from genomic DNA or from RNA
- In any RNA sequencing library there’s an additional step: the RNA conversion in cDNA
- Reverse transcription PCR allows the use of RNA as a template to generate complementary DNA (cDNA)
- cDNA can then serve as template in a variety of downstream applications for RNA studies such as gene expression; therefore, cDNA synthesis is the first step for many protocols in molecular biology
- 5 Steps to Optimal cDNA Synthesis
- Prepare sample
- Maintaining RNA integrity is critical and requires special precautions during extraction, processing, storage, and experimental use
- The main goals of isolation workflows are to stabilize RNA molecules, to inhibit RNases, and to maximize yield with proper storage and extraction methods
- Remove genomic DNA
- Trace amounts of genomic DNA (gDNA) may be co-purified with RNA
- Contaminating gDNA can interfere with reverse transcription and may lead to false positives, higher background, or lower detection in sensitive applications such as RT-qPCR
- The traditional method of gDNA removal is the addition of DNase I to preparations of isolated RNA
- Unfortunately, RNA loss or damage can occur during DNase I inactivation treatment
- Select reverse transcriptase
- Most reverse transcriptases used in molecular biology are derived from the pol gene of avian myeloblastosis “virus” (AMV) or Moloney murine leukemia “virus” (MMLV)
- Prepare reaction mix
- In addition to enzyme and primers, the main reaction components for reverse transcription include RNA template (pre-treated to remove genomic DNA), buffer, dNTPs, DTT, RNase inhibitor, and RNase-free water
- Perform cDNA synthesis
- Reverse transcription reactions involve three main steps: primer annealing, DNA polymerization, and enzyme deactivation
- Prepare sample
- RNA degrades easily because the extra hydroxyl group in the ribose sugar is highly reactive
- To maintain the validity of experimental results, you need cDNA that faithfully represents the template RNA
- Major hurdles in cDNA synthesis reactions include:
- The secondary RNA structures that may slow or even halt reverse transcription
- Degradation of template RNA by the intrinsic RNAase activity of reverse transcriptases (RTs)
- Inhibitors present in RNA samples may reduce polymerization activity of the reverse transcriptase enzymes
- Template degradation and inefficient polymerization result in cDNA of low quality and quantity
- Single-stranded RNA forms hairpin loops and other secondary structures, which can interfere with cDNA synthesis
- Too much RNase activity can degrade template RNA prematurely, which can lower the yield and length of cDNA products
- Substances that bind to RNA and interfere with cDNA synthesis are commonly carried over from RNA sample sources
- A frequent cause of concern among investigators performing quantitative RT-PCR is inaccurate data due to DNA contamination in RNA preparations
- There is no easy remedy and procedures can only reduce the impact of this persistent problem
- Most techniques used for total RNA isolation yield RNA with significant amounts of genomic DNA contamination
- PCR cannot discriminate between cDNA targets synthesized by reverse transcription and genomic DNA contamination
- A known feature of reverse transcriptases is that they tend to produce false second strand cDNA through DNA-dependent DNA polymerase
- PCR amplification has been proven to be the main source of artifacts and base composition bias in the process of library construction, which may lead to misleading or inaccurate conclusions in data analysis
- Sources of PCR Amplification Biases:
- Extremely AT/GC-Rich
- Studies have been indicated that fragments of GC-neutral can be amplified more than GC-rich or AT-rich fragments
- Therefore, the fragments with high AT or very high GC content often have little or no amplification at all
- PCR Cycle
- The higher cycle number can produce significant biases or artifacts in standard amplifications of mixed templates
- Reducing the number of cycles of amplification can decrease (not eliminate) PCR biases and artifacts
- Extremely AT/GC-Rich
- The disadvantage of RT-PCR is that it can lead easily to false-positive results
- A 2011 study reported with high reproducibility the presence of non-specific cDNA amplification with five of the most frequently used reverse transcriptase on three genes in several human cell lines in absence of added primers
- They reported that the presence of RNA contaminants associated with one of the reverse transcriptase studied might contribute to non-specific cDNA synthesis
- This non-specific cDNA synthesis leading to strand-specificity aberrant detection was previously observed in the field of Virology where this approach was used to distinguish between negative and positive-strand “viral” RNA and is known as «false-priming»
- Several mechanisms have been proposed to explain how cDNA synthesis comes from false-priming during the RT reaction, including random priming by contaminating endogenous or exogenous nucleic acids and RNA secondary hairpin structures that can be recognized and extended by the RT enzymes, the so-called «self-priming»
- This false-primed event has been well documented in the field of Virology, in which it is a high source of false-positive results during strand-specific “viral” RNA detection
- It is believed that the origin of primer-independent cDNA synthesis comes from:
- The presence of cellular nucleic acids contaminants like oligo(dT),
- Exogenous (from outside of us) nucleic acid contaminants
- Secondary structured RNAs
- The observation that the amount of background was not the same depending on the RT enzyme raised the possibility that non-specific cDNA synthesis came from the enzyme themselves rather than from the RNA template
- As extensively described in “virus,” they showed that initiation of cDNA synthesis during RT-PCR can occur on human cellular mRNA without addition of any exogenous primers in the RT step
- This can interfere with RT specificity and is a high source of false-positive results, especially in the discrimination of antisense from sense RNA
It is readily apparent that the conversion of RNA to cDNA in order to generate a sequencing library is as fraught with issues relating to contamination, biases, artifact creation, sequencing errors, false-results, etc. as are the prior steps related to “purification” and fragmentation of the RNA. The entire process relies on an enzyme in reverse transcriptase that was obtained synthetically from the pol gene of either the avian sarcoma or murine leukemia “viruses.” Obviously, in order for this to be accurate, it requires the purification and isolation of either of these “viruses” first in order to isolate a specific gene of an enzyme supposedly created from them. As no “virus” has ever been properly purified and isolated, the RT enzyme critical to the creation of the cDNA required to sequence a “virus” is just as hypothetical as the “viruses” being sequenced.
It is well known that the RT-PCR method is prone to false-results, biases, artefacts, and contamination. However, this step is crucial as it supposedly creates the cDNA material used for sequencing. Are we to believe that the numerous steps involved in this genome creation process remain completely contamination/bias/error free when that is obviously not the case as admitted in various sources and studies? Is that even a realistic expectation and outcome knowing the numerous steps involved and the various ways things can, and regularly do, go wrong? Every additional process that the original sample undergoes such as the extraction of the RNA, the fragmentstion of the RNA, and the coversion of the RNA to cDNA, is another step further removed from reality. Every reaction is unobservable, explained hypothetically, and entirely reliant on limited technology and the belief that every step occurs appropriately and accurately free from error. Taking all of the information into consideration, the only logical conclusion that can be made is that the conversion of the RNA into cDNA through RT-PCR is just as hypothetical as the “viruses” said to be sequenced and the enzymes used in their creation.