Why is reverse transcriptase used in PCR?

Why is reverse transcriptase used in PCR?

RT-PCR uses the reverse transcriptase enzyme to convert the RNA material obtained from the sample into complementary DNA (cDNA), which is further amplified using a DNA polymerase and primers to specific biomarker genes. The technology uses fluorescent primers to amplify the genes under investigation.

What are the steps of RT-PCR?

RT-PCR Protocol

1. Experiment process.
2. (1) Primer design. Design and synthesize the primers of the target gene.
3. (2) RNA extraction.
4. (3) Reverse transcription(RNA→cDNA)
5. (4) Real-time PCR.
6. (5) Result analysis.
7. The factors affecting Real-time PCR results.

How does reverse transcriptase work?

When the viral oncogene infects another cell, an enzyme called reverse transcriptase copies the single-stranded genetic material into double-stranded DNA, which is then integrated into the cellular genome.

What is the difference between reverse transcriptase PCR RT-PCR and standard PCR?

RT–PCR is a variation of PCR, or polymerase chain reaction. The two techniques use the same process except that RT–PCR has an added step of reverse transcription of RNA to DNA, or RT, to allow for amplification.

What is the importance of reverse transcription?

Reverse transcription is the key to obtaining the initial DNA (cDNA) which can then be used in a number of applications to further study a gene. RT-PCR (reverse transcription polymerase chain reaction) – Simply put, this is PCR where RNA is the starting material.

How do I perform a reverse transfection with Lipofectamine rnaimax?

If you want to use Lipofectamine RNAiMAX for your cotransfections, perform a reverse transfection with the following modifications: 1: Add 20 ng (for 24-well format) of plasmid DNA to the diluted RNAi duplex.

What is Lipofectamine transfection reagent?

Lipofectamine® Transfection Reagent is a value alternative for transfection of plasmid DNA into eukaryotic cells with a simplified protocol. For optimal results in a wider range of plasmid DNA transfection conditions, we recommend Lipofectamine® LTX Reagent, due to its optimal balance of potency & low-cytotoxicity.

What is the protocol for reverse transfection?

Protocol – Reverse Transfection. Use this procedure to reverse transfect Stealth RNAi or siRNA into mammalian cells in a 24-well format (for other formats, see Scaling Up or Down Transfections). In reverse transfections, the complexes are prepared inside the wells, after which cells and medium are added.

What is M-MLV reverse transcriptase?

M-MLV Reverse Transcriptase is a recombinant DNA polymerase that synthesizes a complementary DNA strand from single-stranded RNA, DNA, or an RNA:DNA hybrid.