Rt pcr

BASIC CONCEPTS ON PCR

1. [email protected] Reverse transcription polymerase chain reaction ( RT-PCR)

2. "The amount of the RNA present in a sample can be quantified by using either fluorescent dye or probe by synthesizing cDNA from RNA using the reverse transcriptase enzyme."

3. PCR Terminology

4. INTRODUCITON:- It is a technique used to monitor the progress of a PCR reaction in real-time. At the same time, a relatively small amount of PCR product (DNA, cDNA or RNA) can be quantified. It is based on the detection of the fluorescence produced by a reporter molecule which increases, as the reaction proceeds. It is also known as a  quantitative polymerase chain reaction ( qPCR ) , which is a laboratory technique of molecular biology based on the  polymerase chain reaction (PCR). qPCR is a powerful technique that allows exponential amplification of DNA sequences. A PCR reaction needs a pair of primers that are complementary to the sequence of interest. Primers are extended by the DNA polymerase.

5. The copies produced after the extension, so-called amplicons , are re-amplified with the same primers leading thus to exponential amplification of the DNA molecules.  After amplification, however, gel electrophoresis is used to analyze the amplified PCR products and this makes conventional PCR time consuming; since the reaction must finish before proceeding with the post-PCR analysis. Real-Time PCR overcomes this problem. The term “real-time” denotes that it can monitor the progress of the amplification when the process is going on in contrast to the conventional PCR method where analysis is possible only after the process is completed.

6. Principle of Real Time PCR From the template RNA, the cDNA is synthesized using the reverse transcriptase. the process is divided into two broad steps; first, reverse transcription, and second, amplification as well as quantification.  As we said, the enzyme governs the process of cDNA synthesize while using probes and primer, the template is amplified and quantified. OR Quantitative  reverse transcription PCR  ( RT - qPCR ) is used when the starting material is RNA. In this method, RNA is first transcribed into complementary DNA ( cDNA ) by  reverse transcriptase  from total RNA or messenger RNA (mRNA). The cDNA is then used as the template for the  qPCR  reaction

7. Depending upon that the RT- qPCR can be performed by two methods: One-step RT-PCR Two-step RT-PCR One-step RT-PCR: In a single tube or single reaction, reverse transcription and amplification are performed (therefore it is named as one-step RT-PCR). It is widely used in repeat quantification assays and high throughput screening due to its high accuracy, specificity, and easy to use & simple set up Two-step RT-PCR: Contrary to the one-step method, in the two-step RT-PCR the reverse transcription and amplification are performed in two separate reaction tubes. That is why this variation is known as two-step RT-PCR. Notably, both reactions have different conditions and ingredients used in it.

8. Process: The procedure of RT- qPCR completed in the following steps, Sample preparation Selection of primers Reaction preparation RT-PCR cyclic condition Strand synthesis

9. Steps of Real Time PCR

10. A.Sample preparation: Instead of DNA, RNA is extracted for the RT-PCR. For extracting the RNA use ready to use RNA extraction kits, it performs better and the yield of the extraction is even good. We have to extract RNA, not DNA. Care must be taken while extraction as RNase present on every possible surface in a lab. RNase is an enzyme cleaves RNA. Here we are extracting total RNA, not mRNA for gene expression study.

11. B.Selection of primers: In the next step select the primer for the experiments. Three types of primers can be used in the reverse transcription PCR. 1.Random primers Random primers are short single-stranded sequences of hexamers or octamers . The random primer binds at the complementary random location on the RNA. It can bind to many types of RNA ( tRNA , rRNA or mRNA) and synthesizes the cDNA .

12. 2.Oligo( dT ) primers The oligo ( dT ) primers are specially designed to amplify the mRNA. As we know that the mRNA has a poly-A tail, the oligo ( dT ) primers only bind to the poly-A tail of mRNA. Hence it is used to amplify entire mDNA into cDNA . It can even amplify smaller mRNAs as well.

13. 3.Sequence-specific primer The sequence-specific primers are commonly utilized in one-step RT-PCR to amplify a gene of interest. The sequences in the sequence-specific primers are complementary to the sequence of our interest therefore, it can’t amplify other gene regions.

14. Components used in the RT- qPCR : The major components are, DNA primers dNTPs Reverse transcriptase enzyme with RNase activity RNase H (if the reverse transcriptase does not have it) DNA polymerase RT- qPCR buffer with RNase inhibitors and PCR enhancers DEPC treated nuclease-free water DNA ligase

15. Temperature conditions for RT- qPCR Here, the denaturation step is not required. The PCR reaction starts with the primer annealing. At the first stage, the primer binds to the template RNA, once it’s done, the reaction is placed for cooling at 4°C for proper binding. New strand synthesis initiates in stage two or second step, afterward, that is followed by the enzyme deactivation in step three. Note:  The length of the primers, the composition of primers, the types of enzyme used in the reaction, and the length of the amplicon decides reaction temperature in each step. Thee different steps of RT-PCR are shown in the figure:

16. Strand synthesis The mechanism of strand synthesis is explained into the figure below

17. PROCESS IN RT-PCR

18. Amplification Denaturation High temperature incubation is used to “melt” double- stranded DNA into single strands and loosen secondary structure in single-stranded DNA. The highest temperature that the DNA polymerase can withstand is typically used (usually 95°C). The denaturation time can be increased if template GC content is high. Annealing During annealing, complementary sequences have an opportunity to hybridize, so an appropriate temperature is used that is based on the calculated melting temperature (Tm) of the primers(5°C below the Tm of the primer).

19. Extension At 70-72°C, the activity of the DNA polymerase is optimal, and primer extension occurs at rates of up to 100 bases per second. When an amplicon in real-time PCR is small, this step is often combined with the annealing step using 60°C as the temperature At 70-72°C, the activity of the DNA polymerase is optimal, and primer extension occurs at rates of up to 100 bases per second. When an amplicon in real-time PCR is small, this step is often combined with the annealing step using 60°C as the temperature.

20. B. Detection The detection is based on fluorescence technology. The specimen is first kept in proper well and subjected to thermal cycle like in the normal PCR. The machine, however, in the Real Time PCR is subjected to tungsten or halogen source that lead to fluoresce the marker added to the sample and the signal is amplified with the amplification of copy number of sample DNA. The emitted signal is detected by an detector and sent to computer after conversion into digital signal that is displayed on screen. The signal can be detected when it comes up the threshold level (lowest detection level of the detector).

21. Fluorescence Markers used in Real Time PCR

22. There are many different markers used in Real Time PCR but the most common of them include: Taqman probe. It is a hydrolysis probe which bear a reporter dye, often fluorescein (FAM) at its 5’ end and a quencher tetramethylrhodamine (TAMRA), attached to the 3’ end of the oligonucleotide . SYBR Green. This is a dye that emits prominent fluorescent signal when it binds at the minor groove of DNA, nonspecifically. Other fluorescent dyes like Ethidium Bromide or Acridine Orange can also be used but SYBR Green is better used for its higher signal intensity.

23. Advantages It has many advantages over the normal PCR: It gives a look in to the reaction that is help to decide which reactions have worked well and which have failed. The efficiency of the reaction can be precisely calculated. There is no need to run the PCR product out on a gel after the reaction as the melt curve analysis serve the purpose. The real-time PCR data can be used to perform truly quantitative analysis of gene expression. In comparison, old fashioned PCR was only ever semi-quantitative at best. Faster than normal PCR. Less complexity at the quantification of sample.etc. Thus, unlike the ordinary preparative PCR, Real Time PCR allows the success of multiple PCR reaction to be determined automatically after only a few cycles, without separate analysis of each reaction, and avoids the problem of “false negatives”.

24. Disadvantages: The method is extremely sensitive, even a small amount of DNA contamination can lead to false results. The method is restricted for some of the assays as higher expertise and experimentation are required to develop new assays.

25. Applications Gene expression analysis Cancer research Drug research Disease diagnosis and management Viral quantification Food testing GMO food Animal and plant breeding Gene copy number

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