![INDEX
PAGE NUMBER TITLE
1 WHAT IS REVERSE TRANSCRIPTION?
2-3 RETROVIRUSES
4-5
MECHANISM OF REVERSE
TRANSCRIPTION IN RETROVIRUSES
6-7 REVERSE TRANSCRIPTASE
8-9
MECHANISM OF REVERSE
TRANSCRIPTION IN TELOMERES
10 TELOMERASE REVERSE TRANSCRIPTASE
11-12 REVERSE TRANSCRIPTION INHIBITORS
13
SIGNIFICANCE OF REVERSE
TRANSCRIPTION
14-15
REVERSE TRANSCRIPTION POLYMERASE
CHAIN REACTION
16-17 STEPS INVOLVED IN RT-PCR
18 TYPES OF RT-PCR
19 ONE STEP RT-PCR
20-21 TWO STEP RT-PCR
22-23 REAL TIME RT-PCR [qPCR]
24 END POINT RT-PCR
25 RT-PCR APPLICATIONS
26 FACTORS AFFECTING RT-PCR
27 ADVANTAGES OF RT-PCR
28 DISADVANTAGES OF RT-PCR
29 REFERENCES](https://image.slidesharecdn.com/rtpcraman-240302160019-4a3c228c/85/RTPCRpresentationAmanNanavatySlideShow-pdf-2-320.jpg)
![SIGNIFICANCE OF
REVERSE
TRANSCRIPTION
•Next-gen sequencing of cDNA
• RT-PCR: RT-PCR [Reverse Transcription - Polymerase Chain
Reaction] is an ef
fi
cient technique used to amplify even minute
quantities of DNA/RNA samples. It is widely used in metagenomics,
metabolomics, ecological diversity studies and species identi
fi
cation.
• Study of Retrovirus Pathology: To understand exact mechanisms of
infection, scientists are studying process of Reverse Transcription in
different retroviruses.
• cDNA labelling: Labelling of cDNA with
fl
uorescent marker (GUS,
GFP, SyBr Green I) is used to elucidate the complete pathway of
genetic info transfer.
• Study of role of Telomerase in Tumours: Recent studies have
shown that unregulated telomerase activity correlates to cancer/
malignancy. Researchers are currently studying the effects of mutation
of TERT gene on normal function.
• In vitro drug studies: The ef
fi
cacy of RT inhibitors such as NRTIs
and Integrase Inhibitors are tested in vitro.
13](https://image.slidesharecdn.com/rtpcraman-240302160019-4a3c228c/85/RTPCRpresentationAmanNanavatySlideShow-pdf-15-320.jpg)
![22
REAL TIME RT-PCR [qPCR]
Real Time RT-PCR is considered as the gold standard for the detection of
RNA viruses (SARS-Cov-2, Zika). It is the most preferred mode of RT-PCR,
often used in quanti
fi
cation and array analysis.
It involves analysis and detection of PCR products in real time using suitable
probes. These probes either bind to the DNA or couple to a quencher moiety
via FRET to produce a detectable
fl
uorescent signal. The probes also confer
higher sensitivity as the instrument will only detect the PCR product if the
probes attach. Real Time RT-PCR is more accurate as it measures
quanti
fi
cation at Exponential phase and continuously measures
fl
uorescence
after every cycle of PCR.
Some of the major probes used are:
TaqMan Probes- Modi
fi
ed oligonucleotides with a quencher at 3’ End
and Fluorescent Probe at 5’ End. TaqMan probes are cleaved during
ampli
fi
cation. They are highly speci
fi
c and generate
fl
uorescence by
FRET coupling. Using TaqMan probes is expensive and challenging.
Scorpion Probes- Scorpion extension on 3’ End binds to
complement on amplicon which opens the scorpion structure. This
prevents FRET and
fl
uorescent signal is then measured.
Molecular beacons- Unlike TaqMan probes, they are not cleaved
during ampli
fi
cation. When beacon hybridises to target, dye and
quencher separate which emits light. They are expensive to
synthesize.
Compared to End Point RT-PCR, it has higher sensitivity and speci
fi
city while
also involving lesser steps. These factors make Real Time PCR the RNA
quanti
fi
cation technique of choice.](https://image.slidesharecdn.com/rtpcraman-240302160019-4a3c228c/85/RTPCRpresentationAmanNanavatySlideShow-pdf-24-320.jpg)
RTPCRpresentationAmanNanavatySlideShow.pdf
- 2. INDEX PAGE NUMBER TITLE 1 WHAT IS REVERSE TRANSCRIPTION? 2-3 RETROVIRUSES 4-5 MECHANISM OF REVERSE TRANSCRIPTION IN RETROVIRUSES 6-7 REVERSE TRANSCRIPTASE 8-9 MECHANISM OF REVERSE TRANSCRIPTION IN TELOMERES 10 TELOMERASE REVERSE TRANSCRIPTASE 11-12 REVERSE TRANSCRIPTION INHIBITORS 13 SIGNIFICANCE OF REVERSE TRANSCRIPTION 14-15 REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION 16-17 STEPS INVOLVED IN RT-PCR 18 TYPES OF RT-PCR 19 ONE STEP RT-PCR 20-21 TWO STEP RT-PCR 22-23 REAL TIME RT-PCR [qPCR] 24 END POINT RT-PCR 25 RT-PCR APPLICATIONS 26 FACTORS AFFECTING RT-PCR 27 ADVANTAGES OF RT-PCR 28 DISADVANTAGES OF RT-PCR 29 REFERENCES
- 3. WHAT IS REVERSE TRANSCRIPTION? Reverse Transcription (RT) is the process through which retroviral DNA is synthesised from RNA via Reverse Transcriptase. In simple terms, it is the synthesis of DNA from a RNA template. It occurs in Retroviruses, Bacterial Retrons, Telomeres and Retrotransposons. Reverse Transcription is used by retroviruses to infect cells by incorporating their genomes into host genomes and by eukaryotes to extend telomere length via telomerase. A common misconception is that reverse transcription only occurs in viruses, but actually it occurs in all eukaryotes and even some bacteria. Eukaryotes use Telomerase to replenish the telomere cap, thereby countering the Hay fl ick Limit. Retrotransposon mobile elements facilitate the replication of viral genomes in infected host cells, allowing for the widespread transcription and translation of the viral genome. Unlike normal transcription, it involves RNA-dependent DNA Polymerase activity as well as DNA- dependent DNA polymerase activity. Ever since its discovery, RT has been extensively studied. RT is used in RT-PCR that is used for the ampli fi cation of both DNA and RNA samples as well as in pathological studies of RNA viruses. 1
- 4. RETROVIRUSES Retroviruses are class VI ssRNA-RT viruses that insert a DNA copy of its own RNA genome into the host cell’s DNA genome by the process of Reverse Transcription. Upon invading the host cell, retroviruses use an enzyme called Reverse Transcriptase that synthesises DNA from a RNA template. They then utilise another enzyme, Integrase to incorporate the newly synthesised DNA into the host genome. After the retroviral DNA has been incorporated into the host genome, the encoded viral genes are transcribed and translated alongside the host’s genes. There are 3 major subfamilies of retroviruses: Oncoretrovirinae- Tumour causing viruses such as HTLV, MLV, Hepatitis C Virus, RSV Spumaretrovirinae- Foamy viruses (benign) that do not cause disease in animals, such as Simian Foamy Virus Lentivirus- Responsible for chronic diseases with long incubation times in animals, such as HIV, BIV, Puma Lentivirus and EIAV Retroviruses have a diameter of around 0.1 um. They have 2 identical single-stranded RNA molecules that are 7-10 kb long, which form a kissing-stem loop structure. The RNA is enclosed by a protective lipid bilayer. Retroviruses encode the following proteins: Protease (pro)- plays a role in proteolytic cleavage during virion maturation Pol- involved in synthesis and integration of viral DNA Env- involved in initial association and entry of virions into host cell gag- main components of capsid, consists of MA and NC domains. Also regulates assembly These viruses have gained the attention of the scienti fi c community due to their role in many notable diseases such as HIV/AIDS, Avian Myeloblastosis, Hepatitis C and Human T-cell Leukaemia. 2
- 5. STRUCTURE OF RETROVIRUSES RETROVIRUS CLASSIFICATION 3
- 6. MECHANISM OF REVERSE TRANSCRIPTION IN RETROVIRUSES Retrotranscription consists of the following steps: 1. Lysyl tRNA, acting as primer, hybridises to Primer Binding Site (PBS) on the viral RNA genome 2. Reverse Transcriptase adds nucleotides that are complementary to U5 and R regions to the 3’ end of primer- cDNA synthesis 3. RNase H degrades U5 and R regions on 5’ end 4. tRNA jumps to 3’ end of viral genome, newly synthesised DNA strands hybridises to complementary R region 5. cDNA extended by nucleotide addition 6. Except for PP sequence, entire viral RNA degraded by RNase H 7. Using PP sequence as primer, second strand of DNA synthesised 8. tRNA primer leaves, causing a jump that results in PBS of second strand to hybridise with complementary PBS of fi rst strand. Formation of dsDNA by strand transfer 9. Both strands extended to form dsDNA copy of viral RNA genome. 10. dsDNA copy integrated into host genome via Integrase 4
- 7. PROCESS OF REVERSE TRANSCRIPTION IN RETROVIRUSES 5
- 8. REVERSE TRANSCRIPTASE Reverse Transcriptase is a RNA-dependant DNA polymerase that was fi rst isolated from retroviruses. It is a multifunctional enzyme that has RNA-dependant DNAP, DNA-dependant DNAP and Integrase activity. Initially believed to be exclusive to retroviruses such as HIV, BIV and Hepatitis B virus, many different kinds of reverse transcriptases have been found in eukaryotes, such as humans, Schizosaccharomyces pombe and Bombyx mori. The structure of RT varies among species, with HIV-1 RT being a heterodimer while M-MLV RT is a single 75kDa monomer. AMV RT consists of 2 subunits, 63 kDa and 95 kDa. HIV-1 Reverse Transcriptase (HIV-RT) is an asymmetric heterodimer consisting of 1 p66 subunit and 1 p51 subunit. The p66 subunit is 66kDa catalytic region that is responsible for the RT activity. On the other hand, p51 subunit is a 51 kDa protein that supports the p66 subunit. Both subunits are derived from the PR- catalysed cleavage of Gag-Pol polyprotein. RT also has ribonuclease H (RNase H) domain that degrades the RNA template upon cDNA synthesis. Integrase domain may either be present or free of RT. Integrase facilitates the entry of synthesised dsDNA copy of viral RNA into the host cell genome. RT has a right-hand structure that comprises of fi ngers (1-85, 118-155), palm regions (86-117, 156- 237), thumb region (237-318) and connection region (319-426); quite similar to most viral nucleic acids polymerases. The nucleic-aid binding cleft is formed by p66 fi ngers, palm, thumb, connection and p66 RNase H domain. p51 subdomains (connection and thumb) form the the fl oor of the binding cleft. 6
- 9. REVERSE TRANSCRIPTASE ACTION 7 HIV REVERSE TRANSCRIPTASE STRUCTURE
- 10. MECHANISM OF REVERSE TRANSCRIPTION IN TELOMERES Telomeres are repetitive DNA sequences present on the ends of the chromosome which shorten after every subsequent replication. To reverse the shortening of telomere cap, telomerase catalyses the addition of CERN (in humans, TTAGGG) to the ends of the chromosome. This nucleic acid sequence is repeated about 100-1000 times. The enzyme telomerase attaches to the end of the chromosome. The enzyme consists of 2 subparts, the catalytic TERT protein and RNA template-containing TR component. First, telomerase adds bases complementary to RNA template to the 3’ end of the overhang. Once the lagging strand is suf fi ciently elongated, DNA Polymerase along with Primase use it as a template to synthesise cDNA to the ends of the chromosome, thereby extending the length of the telomere cap. Telomerase acts on most telomeres without regarding the telomere length, focussing on extending the telomere cap that is shortened on replication. This process prevents ageing and senescence of old cells, while bypassing the Hay fl ick limit. Telomerase confers immortalisation of cells by ensuring that multiple replications do not harm the telomere, that in turn protects the DNA in the chromosome. 8
- 12. TELOMERASE REVERSE TRANSCRIPTASE The Telomerase enzyme is involved in lengthening the telomeres in DNA strands, facilitating cells to exceed the Hay fl ick limit and achieve immortalisation. Its main component, Telomerase Reverse Transcriptase (TERT) is the catalytic subunit that catalyses the addition of nucleotides (TTAGGG seq) to the ends of chromosome’s telomeres. It is a ribonucleoprotein polymerase that is up-regulated in rapidly dividing cells such as embryonic stem cells. TERT confers self-renewal of stem cells to ensure bypassing of the Hay fl ick limit. The pluripotency and multipotency of stem cells is indicated by high levels of TERT. The core human telomerase enzyme consists of 2 main components: 1. TERT Protein- comprises of Reverse Transcriptase Domain (RT), C Terminal extension (CTE), Telomerase Binding Domain (TBRD) and essential N-Terminal Domain (TEN). TERT palm contains aspartic acid residues and TERT fi ngers bind the incoming nucleotides. It contains 1132 Amino Acids and has a molecular weight of 127 kDa. 2. TR Component- comprises of CR4/5, Psuedoknot (contains RNA template) and H/ACA regions 10 CORE HUMAN TELOMERASE ENZYME
- 13. REVERSE TRANSCRIPTION INHIBITORS Telomerase reverse transcriptase has been shown to be inhibited by catechin derivatives, phytochemicals and nucleoside analogues. Some phytochemicals such as Genistein and Circumin have been shown to inhibit telomerase activity by inhibition of mTOR pathway (down regulation of phosphorylation). Derivatives of Retinoic Acid, Nucleoside analogues, Quinilone antibiotics and catechin derivatives are being studied as potential drugs to combat malignancy. Telomerase is also inhibited by well-known Nucleoside Reverse Transcriptase Inhibitors (NRTIs) such as abacavir, tenofovir, zidovudine and stavudine. These NRTIs have demonstrated good potential in vitro, raising the possibility of 100% effective anti-HIV drugs in the future. An area of concern is the potentially lethal side effects of abacavir and stavudine in HIV patients. NRTIs and NtRTIs inhibit RT by competing with natural deoxynucleotides for incorporation into the growing viral DNA chain. Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) such as nevirapine and delavirdine are also effective in inhibiting HIV RT. They work by inducing allosteric changes in retrovirus RT, thereby blocking the process of reverse transcription. Some studies suggest that Telomerase RT is unaffected by NNRTIs. NNRTIs inhibit RT by directly binding to the reverse transcriptase (non-competitive inhibition). Reverse Transcription is also inhibited by drugs that target integration of viral DNA copy, known as Integrase Inhibitors. Some examples of Integrase Inhibitors are bictegravir and elvitegravir. Despite these drugs’ good inhibitory action on RT, retroviruses are able to develop resistance to them in a fairly quick time, rendering the drugs useless after a few generations. This developed resistance has proved to be a stumbling block for treatment of Hepatitis C and HIV/AIDS. 11
- 14. 12 MAJOR REVERSE TRANSCRIPTASE INHIBITORS NRTI MECHANISM
- 15. SIGNIFICANCE OF REVERSE TRANSCRIPTION •Next-gen sequencing of cDNA • RT-PCR: RT-PCR [Reverse Transcription - Polymerase Chain Reaction] is an ef fi cient technique used to amplify even minute quantities of DNA/RNA samples. It is widely used in metagenomics, metabolomics, ecological diversity studies and species identi fi cation. • Study of Retrovirus Pathology: To understand exact mechanisms of infection, scientists are studying process of Reverse Transcription in different retroviruses. • cDNA labelling: Labelling of cDNA with fl uorescent marker (GUS, GFP, SyBr Green I) is used to elucidate the complete pathway of genetic info transfer. • Study of role of Telomerase in Tumours: Recent studies have shown that unregulated telomerase activity correlates to cancer/ malignancy. Researchers are currently studying the effects of mutation of TERT gene on normal function. • In vitro drug studies: The ef fi cacy of RT inhibitors such as NRTIs and Integrase Inhibitors are tested in vitro. 13
- 16. REVERSE TRANSCRIPTION- POLYMERASE CHAIN REACTION Reverse Transcription - Polymerase Chain Reaction (RT-PCR) is an advanced technique used for the ampli fi cation of RNA samples. It combines Reverse Transcriptase with the ampli fi cation of PCR to facilitate better and accurate ampli fi cation of DNA and RNA samples. RT-PCR produces DNA complementary to the RNA sample (cDNA) and then using Polymerase Chain Reaction, the cDNA is exponentially ampli fi ed for RNA quanti fi cation. Reverse Transcriptase catalyses the generation of cDNA from RNA sample and Taq Polymerase is involved in polymerising new DNA strands. The RNA sample serves as template for Reverse Transcriptase. The Taq Polymerase also requires suitable DNA primers, dNTPS, buffers and certain bivalent cations along with the DNA template (cDNA). In modern times, RT-PCR has displaced the time-consuming and inef fi cient method of Northern Blot for RNA quanti fi cation. It has allowed for high ampli fi cation of RNA samples without the need of large sample or post- technique processing. The transcripts of any gene can be practically analysed by RT-PCR. RT-PCR is widely used in many streams, such as Food Process Industries, Cancer Research, Quality Assurance, Drug Design, Pathological Studies, Environmental Monitoring and Genome Modi fi cation 14
- 17. 15 COVID-19 SCREENING FACILITATED BY RT-PCR
- 18. 16 STEPS INVOLVED IN RT-PCR 1. Primer Design: Formulate and synthesize highly gene- speci fi c primers with a length 20-30 nucleotides. The primers must not have a secondary structure, which can interfere in ampli fi cation 2. RNA Extraction: Isolate RNA from sample (environmental, plant, prokaryotic or animal) using suitable technique 3. Reverse Transcription: Perform Reverse Transcription using Reverse Transcriptase to generate cDNA from RNA sample that serves as template 4. PCR: Implement suitable method of PCR (either one step / two step or real time / end point) and suitable fl uorescent probe/dye to amplify cDNA 5. Analysis of Results: Using sophisticated computer programs and softwares, generate graphical analysis of results to determine concentration of RNA sample
- 19. 17 STEPS INVOLVED IN RT-PCR SINGLE THERMAL CYCLE OF RT-PCR
- 20. 18 REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION ONE STEP RT-PCR TWO STEP RT-PCR REAL TIME RT-PCR END POINT RT-PCR
- 21. 19 ONE STEP RT-PCR One Step RT-PCR is a popular method of RT-PCR in which both Reverse Transcription and cDNA ampli fi cation via PCR are carried out in the same test tube. It is a fast and accurate method widely used in COVID diagnostics, high throughput screening and analysis of multiple samples. By carrying out all of the enzymatic reactions in a single test tube, it eliminates the risk of contamination while ensuring better optimisation than two step RT-PCR. Lesser handling corresponds to less time and less variation in results. One step RT-PCR requires only sequence speci fi c primers and is carried out in conditions that support both reverse transcription and polymerase chain reaction activity. Results from one step RT-PCR are more reproducible and accurate. The limitations of one step RT-PCR include its lack of fl exibility, lower sensitivity due to combined reactions and inability to detect multiple genes within a single RNA sample. Another drawback is that it is impossible to optimise the 2 reactions separately.
- 22. 20 TWO STEP RT-PCR Two Step RT-PCR uses 2 test tubes, one for Reverse Transcriptase action and another for RNA ampli fi cation. Unlike One Step RT-PCR, it requires both random nonspeci fi c and sequence-speci fi c primers. It is used in cDNA archiving and Multiplex RNA target detection. Reverse Transcription that generates cDNA using RNA as template is carried out in the fi rst test tube. The cDNA is then transferred to another test tube followed by addition of sequence-speci fi c primer. PCR is carried out to amplify the sample. Due to the transferring of cDNA, the risk of contamination is high and the process is more time consuming than one step RT-PCR. Increased sample handling may inadvertently introduce contamination that skews results, increasing the variability of results. However, two step RT-PCR is well-suited for analysis of multiple genes within a single RNA sample. The separation of 2 reactions allow for enhanced priming fl exibility, fi delity and optimisation of each step, resulting in ef fi cient results. Another advantage is the lack of any multiplexing. Two step RT-PCR is more fl exible and broad-ranged than one step RT-PCR, although its longer time and intensive procedure limit its usage.
- 23. 21
- 24. 22 REAL TIME RT-PCR [qPCR] Real Time RT-PCR is considered as the gold standard for the detection of RNA viruses (SARS-Cov-2, Zika). It is the most preferred mode of RT-PCR, often used in quanti fi cation and array analysis. It involves analysis and detection of PCR products in real time using suitable probes. These probes either bind to the DNA or couple to a quencher moiety via FRET to produce a detectable fl uorescent signal. The probes also confer higher sensitivity as the instrument will only detect the PCR product if the probes attach. Real Time RT-PCR is more accurate as it measures quanti fi cation at Exponential phase and continuously measures fl uorescence after every cycle of PCR. Some of the major probes used are: TaqMan Probes- Modi fi ed oligonucleotides with a quencher at 3’ End and Fluorescent Probe at 5’ End. TaqMan probes are cleaved during ampli fi cation. They are highly speci fi c and generate fl uorescence by FRET coupling. Using TaqMan probes is expensive and challenging. Scorpion Probes- Scorpion extension on 3’ End binds to complement on amplicon which opens the scorpion structure. This prevents FRET and fl uorescent signal is then measured. Molecular beacons- Unlike TaqMan probes, they are not cleaved during ampli fi cation. When beacon hybridises to target, dye and quencher separate which emits light. They are expensive to synthesize. Compared to End Point RT-PCR, it has higher sensitivity and speci fi city while also involving lesser steps. These factors make Real Time PCR the RNA quanti fi cation technique of choice.
- 25. 23
- 26. 24 END POINT RT-PCR End Point RT-PCR utilises fl uorescent dyes to detect gene expression levels, rather than fl uorescent primers. PCR products obtained after process are stained with a suitable fl uorescent dye, the resulting fl uorescence is studied to determine expression levels. Commonly used fl uorescent dyes include Ethidium Bromide (carcinogenic) and SyBr green. Alternatively, scintillation counting and Phosphorimager P32 labelling can be used to detect expression. Unlike Real Time RT-PCR, it only measures the fl uorescence after the completion of PCR, resulting in less accurate results. In this process RNA quanti fi cation is performed ad Lag phase, similar to other traditional techniques. It uses densitometric techniques to quantify visible PCR products. There are 3 methods of End Point RT-PCR, namely: Relative RT-PCR: It is the relative quanti fi cation of PCR products with co-ampli fi cation of a suitable internal control along with the gene of interest. The control is used to normalise the sample. Results expressed as ratio of gene signal to control signal. Comparative RT-PCR: Target RNA must compete with ampli fi cation reagents in a single reaction along with an internal standard/control. The results obtained are then compared with an external RNA standard curve to determine RNA concentration. Most convenient method as it does not need investigator. Competitive RT-PCR: Similar to Comparative RT-PCR in which standard curve is directly generated from the results obtained after PCR. It is used for absolute ampli fi cation and involves a synthetic competitor sequence followed by co-ampli fi cation.
- 27. 25 RT-PCR APPLICATIONS RT-PCR is one of the most widely used laboratory techniques, having other inef fi cient and older methods such Northern Blot and regular PCR. Methods such as qPCR and Real Time PCR have been used in quanti fi cation and ampli fi cation of various RNA samples collected from key sources. RT-PCR is being used in detection of cancer as well as in monitoring patient response to therapy. It is used to detect the transcripts produced by the circulating tumour cells. Scientists use RT-PCR to determine the expression levels of these transcripts in cancer patients. Scientists utilise RT-PCR in gene insertion techniques to insert eukaryotic genes into prokaryotic genomes. Genes from eukaryotes, such as plants or insects are inserted into bacterial genomes (such as Escherichia coli). RT-PCR mediated gene insertion has been used in genetic engineering modi fi cations. Detection of genetic diseases such as Lesch-Nyhan Syndrome by analysing mRNA expression levels of mutated HPRT1 is done by RT- PCR. Gene expression studies often involve RT-PCR techniques. For example, expression of target genes involved in important cellular processes are done using RT-PCR followed by Northern Blot. Gal genes in yeast were studied using this method. RT-PCR has been used in detection of contaminant microbes like molds and yeasts in dairy products (milk, yogurt, etc.) and other foods meant for human consumption. It is a key technique in Quality Assurance programs of Food Industries Genomes of RNA viruses such as HIV, SARS-Cov-2, West Nile Virus, Zika Virus and In fl uenza A virus are studied using extensive RT-PCR.
- 28. FACTORS AFFECTING RT-PCR RT-PCR is affected by: 1. Viral Load of Sample: Results are highly dependant on the Viral Load of the sample. 2. Collection Procedure: The sample collection may in fl uence the PCR products and results. Before performing RT-PCR, the collection method should be chosen carefully in accordance to nature of RNA sample. 3. Nature of Sample: Many times samples from different sources (but same location/person) can have different results. For example, in COVID testing, the results from Upper Respiratory Tract and Lower Respiratory Tract differ. 4. Unsafe transport of PCR products may introduce contamination 5. Storage: Incorrect storage of reagents involved or PCR products can lead to false and erroneous results. After this, the entire process must be repeated, wasting money and time. 6. Primer Bias: Primer Bias due to primer-binding site mismatch can undermine hybridisation, reducing accuracy and correctness of results. Primers must be carefully selected before initiating RT-PCR. 7. Incorrect Interpretation: Sometimes, the results may be wrongly interpreted from the graph/readings by the researcher. RT-PCR should be repeated a few times to minimise variation and ensure accurate and precise results. 8. Varying Probe Dimerisation in different Sample Test Kits 26
- 29. 27 ADV ANTAGES OF RT-PCR 1. Due to its high speci fi city and sensitivity, RT-PCR can give a reliable and accurate diagnosis in just a few hours 2. High con fi dence detection of low copy targets is possible 3. Compared to other diagnostic methods, it requires less time 4. Entire process is carried out in a single test tube 5. Unlike standard PCR, does not need post-analysis gel electrophoresis 6. Lower potential for contamination and hence, less chance of errors 7. Very wide dynamic range as RT-PCR can detect between 1-1011 copy targets 8. Provides both quantitative and qualitative information 9. Tolerates sample RNA degradation as long as primer is intact 10. Easier automation than conventional PCR
- 30. DISADV ANTAGES OF RT-PCR 28 1. Negative RT-PCR tests do not always exclude infection and hence RT-PCR cannot be used as a sole diagnosis tool 2. Requires complex and careful experiment design 3. Higher costs as RT-PCR must be performed by well trained, experienced professionals in clean and secure labs 4. More time-consuming than ELISA 5. Less scalability due to extensive requirements 6. Short window for detection, as it can only detect presence of viral genetic material during infection 7. More intensive than conventional PCR techniques 8. RT-PCR doesn’t indicate past infection and whether patient has developed immunity. Hence, it is not useful in determining development and spread of the virus
- 32. THANK YOU