Reverse transcription-pcr (rt-pcr) Dr. Hani Alhadrami

Transcription

1 Reverse transcription-pcr (rt-pcr) Dr. Hani Alhadrami

2 Overview Several techniques are available to detect and analyse RNA. Examples of these techniques are: Northern blots, Western blots, and RNase protection assay (RPA). Theses techniques are labour-intensive and requires large amount of fully intact RNA.

3 Overview In contrast, newer method based on PCR is less time-consuming, more sensitive and is able to tolerate partially degraded RNA samples. This method is called:

4 Reverse Trasncription PCR (RT-PCR)

5 Invention of RT-PCR The idea of RT-PCR is based upon retroviruses. Retroviruses have an RNA rather than DNA genome. So they must use reverse transcriptase (RT) to covert their RNA genomes into DNA. Human Immunodeficiency Virus (HIV) is an example of a retrovirus.

6 Application of Rt-PCR RT-PCR is used to test RNA extracts from different tissues for the presence of a particular transcript in order to determine the expression pattern of a gene.

7 Principle of Rt-PCR RT-PCR enables RNA to be used as a template for PCR. RT-PCR comprises two steps: 1. The first step is reverse transcription (also called cdna synthesis). 2. The second step is the utilisation of cdna as a template in PCR.

8 Steps of Rt-PCR 1. cdna synthesis: This is the process of converting RNA to DNA using an enzyme called reverse transcriptase. The resulting DNA is complementary to the RNA template, so it is called complementary DNA (cdna).

9 Steps of Rt-PCR 2. The use of cdna as a template in PCR:

10 Steps of Rt-PCR 2. The use of cdna as a template in PCR: Once the cdna is synthesised from step-1, the PCR primers and Taq polymerase are added and the experiment proceeds exactly as in the standard PCR technique. The amount of PCR product is indicative of the starting amount of input cdna (and by interference, RNA that it was generated from).

11 Steps of Rt-PCR RT-PCR can be performed in two steps, with an RT reaction occurring first, then using that product as a template in a PCR reaction. Alternatively, RT-PCR can be carried out in one step with both reaction occurring in one tube.

12 Ingredients of Rt-PCR The reaction tube of RT-PCR contains the following: Reverse transcriptase: the enzyme catalyses the reverse transcription reaction. There are several commercially available enzymes usually isolated from retroviruses. Buffer. dntps (like those used in PCR), and they will be incorporated into the newly synthesised cdna strand. Primers: which are required to synthesis cdna. RTPCR requires only one primer because only one strand of cdna is made.

13 Primers categories Primers fall into three categories: Oligo-dT, randomers (hexamers) and gene specific. 1. Oligo-dT primers: will specifically anneal to polya tails found on most eukaryotic mrnas. This type of primers cannot be used with prokaryotic RNA. 2. Gene Specific primers: cannot be used if we want to prime cdna synthesis from all the RNAs in the cell. 3. Random hexamers primers: have the ability to anneal to all types of RNA without knowledge of sequence. They are a pool of primers designed to represent all possible combinations of sixbase pair stretches. This design allows for the primers to bind to all RNA sequences.

14 Crucial Aspect Controls RT-PCR experiment DNA contamination must be avoid in RT-PCR experiment. If DNA still present in the RNA sample, it can be amplified by the gene specific primers that amplify the cdna leading to inaccurate quantification of mrna levels.

15 Crucial Aspect Controls RT-PCR experiment To avoid DNA contamination, two approaches can be used: 1. Treat the sample with DNase. 2. Use in the reaction a negative control which dose not contain reverse transcriptase enzyme (-RT). If the DNA is effectively eliminated from the starting material (RNA sample), amplification using -RT cdna template should show no product. The corresponding +RT cdna template should result in a product.

16 THANK YOU REFERENCE: MOLECULAR BIOLOGY TECHNIQUES: A CLASSROOM LABORATORY MAUAL