Difference between PCR and RNA-seq

In modern biological research, it is inevitable to detect the expression of a certain gene or a certain DNA sequence, and RNA-Seq and PCR techniques have come into being, both of which can detect the above mentioned as well as characterize transcripts, transcriptome research, and so on. Although both can be used to analyze RNA expression, they are significantly different in principle, application, advantages and disadvantages.

In this paper, we will explore the main differences between RNA-seq and PCR, and analyze when it is more appropriate to use which technique in relation to specific application scenarios.

Principle of PCR and RNA-seq

The basic principle of RNA sequencing is to extract total RNA from tissues or cell samples, reverse transcribe it into cDNA, and then sequence the cDNA using a high-throughput sequencing platform.RNA-seq is able to comprehensively capture all the transcripts in the samples, including known and unknown genes, as well as transcriptional variants in different splicing forms, and then computationally analyze them to obtain information about the expression levels of the individual genes and transcripts. The expression level of each gene and the transcript information can be analyzed computationally.

PCR is a technique for rapid and large-scale replication of specific DNA fragments in vitro. Based on the amplification principle of DNA semi-reserved replication, specific DNA sequences are rapidly amplified by thermal cycle reaction. For the detection of RNA samples, it is usually necessary to convert RNA into cDNA by RT-PCR and then PCR amplification. PCR needs to detect the expression of specific genes quantitatively or qualitatively by designing specific primers.

The development of the PCR system and its applications (Zhu H et al.,2020).

Process of PCR and RNA-seq

The process of RNA-seq is relatively complex, and general laboratories cannot complete it independently. They need to extract samples and send them to the company such as CD Genomics for sequencing. Please refer to the specific process: "Overview of RNA Sequencing Techniques".

PCR reaction is very simple : (1) high temperature (95 °C) denaturation template; (2) Design specific primers and template complementary pairing for artificial annealing (55 °C); (3) The target chain was synthesized by heat-resistant DNA polymerase at 72 °C. These steps are very simple and are the entry-level operations of each researcher.

The PCR instrument and reagents have also been mass-produced. But there are some precautions : (1) pay attention to prevent DNA contamination, so as not to produce false positives; (2) Do not use excessive reagents; (3) After adding all the reagents, gently blow with the tip of the gun several times to ensure full mixing; (4) Remember to use positive and negative controls; (5) After the completion of PCR, the products should be detected by electrophoresis on the same day, so as not to dissipate the reactants; (6) The use of high-fidelity enzyme amplification longer, high GC content and easy to produce secondary structure of the template.

Classification

The development of RNA-seq is very rapid, and there are many different techniques for different purposes, such as Total RNA-Seq, Bulk RNA-seq, scRNA-seq, Spatial RNA-seq, Iso-Seq, Small RNA-Seq, and Targeted RNA-seq, and so on, which can meet our different needs.

We have a wide range of techniques to meet our different needs, and we are free to choose a certain technique to achieve the results we want quickly, regardless of the purpose of the experiment. Specific RNA-seq techniques and their uses can be found in the following pages: "Overview of RNA Sequencing Techniques".

Since the development of PCR in 1985, a variety of techniques have been developed, the basic principles of which are the same, but still different.

• Ordinary PCR: the most commonly used PCR technology, but which field, as long as the study of a particular gene, know its specific sequence, it is necessary to PCR will be amplified in the subsequent research, such as plants through the construction of vectors to achieve a certain gene overexpression of plant reproduction; animals, the same study of a certain gene also need to be amplified first by PCR. It is mainly used for the amplification and detection of DNA molecules.
• qPCR: The target gene is labeled by a fluorescent probe during the extension of the reaction and the fluorescent signal is collected in real time. The collected data includes three parameters: fluorescent signal, Ct value and the starting concentration of the target gene, and the relationship between them is calculated to determine the copy number of the target gene. However, since the absolute quantitative results of qPCR are too dependent on the Ct value and the standard curve, the results are only relative, and qPCR may not be able to detect the actual expression of low-copy gene molecules. There are two commonly used qPCR methods: TaqMan probe method and SYBR Green method. It generally monitors the content of DNA molecules or cDNA molecules in real time.
• dPCR: Absolute quantitative PCR, the basic principle is to evenly divide the DNA or RNA samples into many tiny reaction units (nano-levels), and amplify the target sequences by single-molecule template PCR in each reaction unit and analyze it by counting the fluorescence detection value. The presence or absence of the target molecule can be determined by digitally counting the positive and negative results of the reaction units, which are independent of standard curves and multiple gradient standards of one concentration, and allow direct detection of the original concentration accounted for in the sample for accurate quantification.
• RT-PCR: RNA molecules are reverse transcribed into cDNA, which is then amplified by PCR. Generally, this reaction is required after RNA extraction and purification in the RNA-seq process in order to build libraries for sequencing.
• RT-qPCR: A combination of qPCR and RT-PCR.

Different PCR techniques (Zhu H et al.,2020).

Data Scope and Cost

A significant advantage of RNA-seq is its wide range of data. It can not only detect known genes, but also find new transcripts, gene fusion, alternative splicing and other transcriptome-level mutation events. In addition, RNA-seq can analyze a variety of RNA molecules in complex samples, including mRNA, miRNA, lncRNA, and small RNA.

However, the cost of RNA-seq is high, requiring expensive sequencing equipment and reagents as well as a powerful computing platform for data processing and analysis. Its data analysis usually requires the use of complex bioinformatics tools, which requires a long time for data cleaning, alignment and quantitative analysis. For complex fusion genes and alternative splicing events, more algorithm support is needed. Moreover, the amount of data generated by RNA-seq is very large, and data storage and processing are also very complicated.

PCR has a more limited scope because it relies mainly on pre-designed primers, so it can only detect specific genes or DNA sequences. PCR is far less abundant than RNA-seq in terms of data volume. It is usually used to verify the expression of known genes and cannot reveal the information of the whole transcriptome. Moreover, its quantitative accuracy is usually affected by factors such as primer design, reaction conditions, and amplification efficiency. Especially when dealing with complex samples, the quantitative results of PCR may be affected and inaccurate.

However, the cost of PCR is low, and the cost of reagents, equipment and time required for PCR reaction is much lower than that of RNA-seq. It is generally possible to complete the process from RNA extraction to final analysis usually in a few hours or one or two days, and its data analysis is also very simple, the analysis process is intuitive and does not require complex calculations. This also shows that PCR is suitable for small-scale, rapid gene expression analysis, and has high sensitivity and accuracy. Especially when resources are limited or some genes need to be quickly verified, PCR is a cost-effective choice.

Key Applications and Advantages

RNA-seq is suitable for those scenarios that require genome-wide and transcriptome-wide analysis. It can provide comprehensive and in-depth information, which is suitable for the discovery of gene expression profiles, the study of gene regulatory mechanisms, the discovery of new transcripts, and DEGs analysis. RNA-seq is especially needed when exploring unknown areas such as new biomarkers, disease mechanisms, gene variants, etc. It is also suitable for high-throughput screening of complex samples, such as cancer tissues, samples from different developmental stages, etc. To learn more about the specific applications of RNA-seq, please refer to:

PCR is suitable for small-scale, target-specific gene expression validation. It is often used for qualitative or quantitative analysis of specific genes or transcripts, especially when studying certain known genes. For example, PCR plays an important role in gene function validation, clinical screening, and mutation detection. Due to its low cost, high sensitivity and rapidity, PCR is more advantageous in some rapid tests and small-scale experiments.

In addition to the several traditional PCRs we introduced above, nowadays a variety of different new PCR methods have been developed based on traditional PCR, which can be applied in different fields of detection.

• Amplification of specific genes: There are many criteria to be considered for the development of therapeutic antibodies against a target antigen, such as target selectivity, low risk of immunogenicity, low non-specific binding, and minimal exploitable propensity. In vertebrate immune response B cells occupy an important position, but the genes expressed by B cells are often unknown i.e. not predictable due to the VDJ properties and the two chains of VL and VH, so a series of primers need to be designed to amplify the B cell VL and VH genes in order to capture the diversity of the immune response. For this reason, it is difficult to develop therapeutic antibodies against B cells. The researchers developed RNase H2-dependent PCR (rh-PCR) to amplify cloned antigen-specific antibodies from immunoreactive individual B cells. rNase H2 cleaves at the side nucleotide only when correct base pairing of the primer-template occurs. rh-PCR amplification is highly specific and increases cloning of low-abundance target genes as well as reduces primer dimer formation. Because the enzyme is thermally stable (50°C-75°C) and least active at room temperature, it can be added directly to PCR mixtures and gives the PCR a strict "hot start" function. Crucially, rh-PCR can be combined with NGS to detect different information about the sequence from a large number of expressed homologous antibodies. VL/VH fragments can be amplified more efficiently from B cells by rh-PCR (Crissman J et al., 2020).
• Differentiating between species: Amoebiasis caused by E. ameba Entamoeba histolytica is a serious parasitic disease that affects an estimated 500 million people worldwide. There are more than 40 species in the genus, eight of which infect humans, but four (E. dispar, Entamoeba histolytica, E. bangladeshi and E. moshkovskii) are morphologically indistinguishable (false positives on microscopic examination), with some success with ELISAs, but qPCR is more successful than ELISAs. qPCR is more sensitive than ELISAs. The researchers developed a new quadruplex real-time PCR method based on hydrolyzed probes for the simultaneous detection of these four parasites, which can be used in the clinic. The four different parasites were identified by tetraplex RT- PCR of selected fecal and liver aspirate samples associated with clinical amoebiasis. Because tetraplex RT- PCR designs unique primers for the four species, it is possible to detect mixtures that distinguish the four species, and distinguishing between the different species in the clinic helps us develop targeted drugs for treatment. Crucially, tetraplex RT- PCR can also be used for routine PCR, allowing for specific analysis even when a qPCR instrument is not available (Ali IKM et al., 2020).
• Detection of the virus: Since the outbreak of the new coronavirus (COVID-19) has caused great damage all over the world, it is essential that we have a rapid and accurate method of detecting the new coronavirus in order to isolate infected people. Conventional PCR was the preferred method to begin with, but we needed to develop a POC device for RT-qPCR that could be portable. The researchers used the MEDIC-PCR, which uses a disposable PCR chip to perform real-time RT-qPCR, accompanied by a miniature, reliable thermocycler and a sensitive fluorescence measurement system, which allows us to go from a sample (1 μl is sufficient) to the final result in only 15 minutes, thus allowing us to rapidly and accurately detect neoconavirus (COVID-19). This enabled us to rapidly and accurately detect COVID-19. In order to test its accuracy and sensitivity, MEDIC-PCR was used to detect different concentrations of SARS-CoV-2 viral RNA in real time (50, 500, 5000, and 50,000 copies per μL) and to use the SARS-CoV-2 reference RNA as a positive control. MEDIC-PCR is 100% efficient in detecting different concentrations of RNA and its plotted standard curve is comparable to that of conventional PCR, but it can detect COVID-19 in less than 15 min.Using it in clinical trials, 192 clinical samples (comprising 89 positives and 103 negatives) were tested using MEDIC-PCR, resulting in 84 Using MEDIC-PCR in a clinical trial with 192 clinical samples (89 positives and 103 negatives), there were 84 "true positives" and 101 "true negatives", but comparing the results to those of conventional PCR, the accuracy was similar, but the sensitivity was higher and the time was greatly reduced (Shrestha K et al., 2023).
• Identification of pathogens: Malaria is a highly prevalent disease in the Greater Mekong Region, of which Anopheles dirus complex is one of the most important vectors, with different interspecies varieties having different transmission capabilities. In order to inhibit the transmission of this disease, we need to identify the different complexes among the species accurately. Currently, the most used method is AS-PCR (polymerase chain reaction specific for the Dirus allele in the ITS2 region), but it cannot accurately recognize the different complexes in the interspecies, which is non-specific. The researchers modified AS-PCR by shortening the thermal cycling time and designing a primer that specifically recognizes An. DiCSIP is different from AS-PCR, which uses forward primer D-U and specific reverse primer D-AC. The forward primer was designed as DiCSIP-Uni-Fwd and the specific reverse primers were DiCSIP-Rev-F, D-D, D-B, and DiCSIP-Rev- AC, which were identified by single PCR, and shown to recognize five species of An. Dirus, and then used in multiple PCR reaction (one additional primer was added). used in a multiplex PCR reaction (with the addition of a new forward primer DiCSIP-Fwd-C) DiCSIP was able to recognize five homologous species of An. Dirus. DiCSIP can recognize the five homologous species of An. Dirus, and only requires a concentration of 10mg to 0.1ng to generate amplicons accurately and sensitively (Saeung M et al., 2024).
• Detection of microorganisms: Complicated urinary tract infection (cUTI) is a complex disease for which there is no cure. cUTI is caused by PMO and FO, so the main test, urine culture (UC), is not very accurate due to the long incubation period, the fact that some microorganisms have an incubation period or don't grow or multiply in UC, and the complex interactions between the microorganisms. The results of cUTI are not very accurate. Therefore, it is very important for us to develop an accurate detection of cUTI. The researchers attempted to detect cUTI by PVR. nearly 36,600 clinical samples of cUTI were selected and tested by UC and PCR, respectively, with the final results being 33.9% positive by UC and 52.3% positive by PCR (18.4% difference, p < 0.01). The patients who tested negative for UC were also tested for PCR, and it was found that 20.4% of them showed PCR positivity, while only 1.9% of the PCR negatives were detected as UC+. Testing for microorganisms in UC+ and PCR+ showed that 96.4% of UC+ specimens had one organism detected, 3.6% had 2 organisms detected, and none of the more than 2 were detected. However, 1, 2, 3, 4 or even more organisms were detected in 53.8%, 24.8%, 11.7% and 9.6% of PCR+ specimens, respectively, with significant differences. When PMO was detected in UC+ and PCR+, 7.0% of PMO was detected in 3.6% of UC+, whereas 70.8% of PMO was detected in 46.2% of PCR+. Not only was the difference in the detection of PMO (42.6%) significant, but the number of organisms detected (63.8%) was significant as well (p < 0.01). FO detection rate: 0.7% for UC+ compared to 31.3% for PCR+ specimens. A total of 45 microorganisms were detected by PCR for cUTI, while the majority (90%) of microorganisms detected by UC were also detected by PCR, but UC only detected 31.9% of PCR+. If the PCR plate size is increased, then all the results such as the number of organisms, PMO and FO detection rate will be significantly increased to be able to adequately detect pathogens by cUTI (Hao X et al., 2023).
• Identification of mutations: Pancreatic cancer is an aggressive malignant tumor that has a poor prognosis despite surgical treatment.PDAC is a common type of pancreatic cancer.Literature has reported that most of the development of PDAC is caused by snp of the KRAS gene.Detecting mutations in the KRAS gene can help us to identify recurrence as well as the prognostic response to PDAC.Previously, detection of mutations in the KRAS gene has been done by liquid biopsy. but its sensitivity and accuracy are limited. The researchers developed an oligoribonucleotide interference PCR (ORNi-PCR) coupled with ddPCR to detect KRAS gene snp. The ORNs of ORNi-PCR were designed to target KRAS mutations, which can specifically bind to the KARS WT sequence, thus enriching for snp at the G12/G13 position, and then optimize the experimental conditions to specifically amplify snp based on the enriched snp. Then RT-PCR or ddPCR was performed by double-labeled probes targeting KRAS G12X and G13X mutations. ddPCR was performed after ORNi-PCR compared to ddPCR without ORNi-PCR (only one positive droplet was detected). The monitoring data (113 positive droplets detected) were significantly increased, and the higher the number of cycles of ORNi-PCR, the higher the ethical positivity rate of ddPCR detection. tens of thousands of data showed that ORNi-PCR followed by ddPCR and then detecting the KRAS mutant DNA in cfDNA was more sensitive and accurate than either direct ddPCR alone or ddPCR after conventional PCR (Fujita H et al., 2024).

However, usually the RNA-seq results need PCR, i.e., a combination of the two.

• Liver cancer is a kind of cancer with high morbidity and mortality rate in China, and the postoperative metastatic events and recurrence rate of liver cancer are also very high, so we need clinical means to effectively and accurately predict the condition of liver cancer and monitor the postoperative treatment effect and metastasis in real time, and liquid biopsy is more often used nowadays because of its high sensitivity, non-invasiveness and dynamics, e.g., human plasma, and the Sc RNA-seq can isolate different cells of different origins in plasma and perform cell-specific RNA-seq to monitor the disease. For example, in human plasma, Sc RNA-seq can isolate plasma cells of different origins and perform cell-specific RNA-seq to monitor the disease. The researchers used Sc RNA-seq in conjunction with digital PCR to detect hepatocellular carcinoma cells and develop new tumor markers. Sc RNA-seq was performed on approximately 17,100+ cells from 4 HCC patients, including HCC tumor cells and healthy normal tissue cells, which identified previously published hepatocyte-specific transcripts as well as new transcripts. CELSIG assessment of specific genes resulted in significantly higher blood CELSIG in HCC cells compared to non-HCC cells, while their CELSIG scores decreased rapidly after 72 h postoperatively, suggesting that cell-specific gene tags identified by Sc RNA-seq can be used for cancer prognostication. To verify the accuracy of the results identified by Sc RNA-seq, the researchers used dPCR to quantify seven members of each of the 39 hepatocyte-specific genes: TF, FGL1, HPD, ORM2, GSTA1, ALDOB, and KNG1. The results of the dPCR were consistent with the expression of the transcripts detected by Sc RNA-seq, and demonstrated that FGL1, which was highly expressed in patients with HCC After surgery, it was significantly decreased, while it was almost not expressed in non-HCC patients, so FGL1 can be used as a prognostic marker, and the use of dPCR can be faster and more efficient to detect FGL1 (Vong JSL et al., 2021).

More applications, refer to "Overview of RNA Sequencing Applications"

Summary

Summarizes the key differences between PCR and RNA-seq

References:

1. Crissman J, Lin Y, Separa K, Duquette M, Cohen M, Velasquez C, Cujec T. "RNase H-dependent PCR enables highly specific amplification of antibody variable domains from single B-cells." PLoS One. 2020;15(11):e0241803. doi: 10.1371/journal.pone.0241803
2. Ali IKM, Roy S. "A Real-Time PCR Assay for Simultaneous Detection and Differentiation of Four Common Entamoeba Species That Infect Humans."J Clin Microbiol. 2020;59(1):e01986-20. doi: 10.1128/JCM.01986-20
3. Shrestha K, Kim S, Han J, Florez GM, Truong H, Hoang T, Parajuli S, Am T, Kim B, Jung Y, Abafogi AT, Lee Y, Song SH, Lee J, Park S, Kang M, Huh HJ, Cho G, Lee LP. "Mobile Efficient Diagnostics of Infectious Diseases via On-Chip RT-qPCR: MEDIC-PCR." Adv Sci (Weinh). 2023;10(28):e2302072. doi: 10.1002/advs.202302072
4. Saeung M, Pengon J, Pethrak C, Thaiudomsup S, Lhaosudto S, Saeung A, Manguin S, Chareonviriyaphap T, Jupatanakul N. "Dirus complex species identification PCR (DiCSIP) improves the identification of Anopheles dirus complex from the Greater Mekong Subregion." Parasit Vectors.2024;17(1):260. doi: 10.1186/s13071-024-06321-6
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6. Fujita H, Fujita T, Ishido K, Hakamada K, Fujii H. "Oligoribonucleotide interference-PCR-based methods for the sensitive and accurate detection of KRAS mutations."Biol Methods Protoc. 2024;9(1):bpae071. doi: 10.1093/biomethods/bpae071
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