Reverse Transcription

Reverse transcription of RNA, which refers to the conversion of the RNA template into its complimentary DNA strand (cDNA) is an essential step in the analysis of gene transcripts.
cDNA can be sequenced, cloned and applied to estimate the copy number of specific genes in order to characterize and to validate gene expression.

1. Reverse transcription FROM RNA TO cDNA

2. REVERSE TRANSCRIPTION OF RNA Reverse transcription of RNA, which refers to the conversion of the RNA template into its complimentary DNA strand (cDNA) is an essential step in the analysis of gene transcripts. cDNA can be sequenced, cloned and applied to estimate the copy number of specific genes in order to characterize and to validate gene expression.

3. REVERSE TRANSCRIPTASES Reverse Transcriptases are RNA dependent DNA polymerases which bind to RNA templates and convert them to their complimentary DNA sequence in the presence of dNTPS and other essential cofactors.

4. REVERSE TRANSCRIPTASES IN VIRUSES The enzyme Reverse Transcriptase was first reported by Dr. Howard Temin. It was discovered in reverse transcribing viruses. These viruses contain a genome which is composed of RNA but which is reverse transcribed to DNA in the host cells.

5. RNA TEMPLATE The RNA template comprises the messenger RNA. RNA is single stranded and tends to form secondary structures via Watson-Crick base pairing. Heating the template is essential in order for it to be linearized prior to annealing of the primers.

6. PRIMERS Three kinds of primers: Oligo dTs are synthetic DNA strands which can be represented as a sequence of ‘T’ nucleotides as in 5’ – G T T T T T T T T T T T T T T T T -3’ Gene specific primers can be designed to anneal to a specific gene. Random hexamers consist of a ten bases which anneal randomly to RNA molecules.

7. REVERSE TRANSCRIPTASES RNA dependent RNA polymerase is derived from reverse transcribing viruses. Mooloney Murine Leukemia Virus. Avian Myeblastoma Virus (AMV).

8. THE PROCESS REVERSE TRANSCRIPTION IN VITRO

9. THE PROCESS OF RT-PCR Linearization of RNA template. Annealing of Oligonucleotides. Reverse transcription by Reverse Transcriptase. Removal of mismatches by RNase H. Mismatch repair by Klenow Fragment DNA Polymerase. Downstream processing of cDNA.

10. LINEARIZING THE RNA TEMPLATE 50 o C

11. ANNEALING OF THE PRIMERS

12. REVERSE TRANSCRIPTION

13. REMOVAL OF MISMATCHES 5’- G U A U G C U G C G C U G G G – 3’ 5 ’- C A T A C G A C G C G A C C C – 3’ 5’- G A G A G G C G G C C – 3’ 5’- C A T A C T A G G C G A G C C – 3’

14. GAP FILLING BY DNA POLYMERASE 5’- A C G A G G C G G C C – 3’ 5’- T T G T C T T C C G C T C G G – 3’ 5’- A A C A G A A G G C G A G C C – 3’ 5 ’- T TG T C T T C C G C T C G G – 3’

15. AMPLIFICATION OF cDNA VIA PCR THE cDNA strand obtained via RT can be subjected to downstream processing via: Quantitative Real Time PCR to determine copy number. PCR to amplify specific genes. Cloning to express specific genes in heterologous systems.

16. Other kinds of R everse T ranscription Random Amplification of cDNA ends or RACE-PCR can be applied to reverse transcripts which are extremely long or which encode genes whose complete sequences are unknown.

17. SUMMARY Reverse Transcription. In vitro reverse transcription. Quantification of expression levels.

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