Introduction of RT PCR

1. Rt -PCR Course: Fundamental of genetic engineering and biotechnology Course code: GEB3 2 Course Instructor: Dr. Mohiuddin Kabir Presented by: Bitali Islam Nilmoni Bushra Mohammad Nabil Hossain Md. Shabab Mehebub

2. Intro duc tion of RT-PCR RT PCR - Reverse transcription polymerase Chain reaction . Reverse transcription polymerase chain reaction ( RT-PCR ) is one of many variants of polymerase chain reaction (PCR). It was introduced in 1977 . The discovery of  reverse transcriptase  during the study of viral replication of genetic material led to the development of RT-PCR. RT-PCR is used to qualitatively detect gene expression through creation of complementary DNA ( cDNA ) transcripts from RNA.\ This technique is commonly used in molecular biology to detect RNA expression.

3. V/S RT-PCR PCR

4. RT -PCR Principles There are several types of RT PCR Principles . One step RT PCR RT-PCR can also be carried out as one-step RT-PCR in which all reaction components are mixed in one tube prior to initiation of the reaction. One-step RT-PCR offers simplicity and convenience and minimizes the possibility for contamination. T he resulting cDNA cannot be used for detecting multiple messages from a single RNA sample as in two-step RT-PCR. Two step RT PCR Traditionally, RT-PCR involves two steps: the RT reaction and PCR amplification. RNA is first reverse transcribed into complementary DNA ( cDNA ) using an enzyme, reverse transcriptase. The resulting cDNA is used as templates for subsequent PCR amplification using primers specific for one or more genes.

5. RT -PCR Principles Quantification of RT-PCR products can largely be divided into two categories: end-point and real-time . End-point RT-PCR : measurement approaches of end-point RT-PCR detect gene expression levels by the use of fluorescent dyes like   ethidium bromide ,  Phosphorus-32  labeling of PCR products using phosphorimager . End-point RT-PCR is commonly achieved using three different methods: relative, competitive and comparative. Real-time RT-PCR : the analysis and detection of PCR products in real-time has consequently led to the widespread adoption of real-time RT-PCR for the analysis of gene expression. There are four different fluorescent DNA probes for the detection of PCR products:  SYBR Green ,  TaqMan ,  Molecular Beacons , and Scorpions . All of these probes allow the detection of PCR products by generating a fluorescent signal.

6. Method of RT PCR

7. Double strand cDNA AAAAA TTTTT RT AAAAA TTTTT RT RT AAAAA TTTTT Oligo dT primer is bound to mRNA Reverse transcriptase (RT) copies first cDNA strand Reverse transcriptase digests and displaces mRNA and copies second strand of cDNA Conversion of mRNA to cDNA by Reverse Transcription

8. A. Double strand DNA B. Denature 96 º 50 º C. Anneal primers 50 º D. Polymerase binds 72 º Taq Taq

9. 72 º Taq Taq E. Copy strands 1 2 3 4 F. Denature 96 º First round of cDNA synthesis (4 strands) Taq Taq

10. 1 2 3 4 50 º G. Anneal primers

11. 1 2 3 4 Taq Taq Taq Taq 72 º H. Polymerase binds

12. 1 2 3 4 Taq Taq Taq Taq I. Copy strands 72 º Second round of cDNA synthesis (8 strands)

13. 1 2 3 4 J. Denature at 96 º Anneal primers at 50 º

14. 1 2 3 4 72 º K. Bind polymerase (not shown) and copy strands Third round of cDNA synthesis (16 strands)

15. 1 2 3 4 L. Denature at 96 º Anneal primers at 50 º

16. 1 2 3 4 M. Copy strands at 72º Fourth round of cDNA synthesis (32 strands) 72 º

17. 1 2 3 4 cDNA strands (32) are now shown as lines

18. 1 2 3 4 After 5 rounds there are 32 double strands of which 24 (75%) are are same size

19. The exponential amplification via reverse transcription polymerase chain reaction provides for a highly sensitive technique in which a very low copy number of RNA molecules can be detected. RT-PCR is widely used in the diagnosis of genetic diseases and, semiquantitatively , in the determination of the abundance of specific different RNA molecules within a cell or tissue as a measure of gene expression. Research methods- For example, Lin et al. used qRT -PCR to measure expression of Gal genes in yeast cells. Gene Insertion- RT-PCR can also be very useful in the insertion of eukaryotic genes into prokaryotes. RT-PCR is commonly used in studying the genomes of viruses whose genomes are composed of RNA, such as Influenzavirus A and retroviruses like HIV. Genetic Disease Diagnosis- RT-PCR can be used to diagnose genetic disease such as Lesch–Nyhan syndrome. Also can be used as a test for bird flu- H7N9. Cancer Detection- Scientists are working on ways to use RT-PCR in cancer detection to help improve prognosis, and monitor response to therapy RT-PCR is commonly used in studying the genomes of viruses whose genomes are composed of RNA, such as Influenzavirus A and retroviruses like HIV. Application of RT-PCR

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