Transcriptome analysis is proving its immense importance day by day as it RNAs are crucial to unterstand the dynamic nature of cellular events. Since RNAs are not stable, they are reverse transcribed to cDNA and the cDNAs are sequence instead of the RNAs to get the sequence of RNAs. But the a mammoth obstacle of this procedure is the incomplete synthesis of cDNA.
There are several reasons why the first strand of cDNA is not fully transcribed during the reverse transcription. Here some of the causes and the ways of overcoming those difficulties will be discussed.
1) Problem: inefficiency in the synthesis of the first cDNA strand
Cause: due to inefficiency of reverse transcriptase enzymes.
Solution: Addition of trehalose.
Mechanism: trehalose gives heat resistance to enzyme including the reverse transcriptional enzyme. From this discovery, it became possible to induce reactions using the reverse transcriptional enzyme with 60°C instead of 42°C as was previously the norm. Under 60°C, the template RNAs' secondary structure gets weaker, and the area with stable secondary structure, which often exists in non-coding 5' end mRNAs, becomes possible to be transcribed in reverse in a very efficient manner.
2) Poblem: absence of actual 3' end of cDNA (i.e. 5' end of the mRNA)
Cause: inefficiency of reverse transcriptase
Solution: Cap-trapper method.
Mechanism: biotinylationof the cap-structure that is specific to mRNA of eukaryotic organisms followed by selective extraction of the biotinylated cap structure of non full-length cDNA (enzymatic degradation of ss mRNA ). The remaining full-length cDNA containing a biotinylated cap can be caught by using magnetic beads coated with streptavidin. The selected full-length cDNA can then be eluted by alkaline treatment. Synthesizing the second strand from the 1st strand cDNA (which became single stranded when separated from the beads) makes it possible to obtain the full-length cDNA selectively.
There are several reasons why the first strand of cDNA is not fully transcribed during the reverse transcription. Here some of the causes and the ways of overcoming those difficulties will be discussed.
1) Problem: inefficiency in the synthesis of the first cDNA strand
Cause: due to inefficiency of reverse transcriptase enzymes.
Solution: Addition of trehalose.
Mechanism: trehalose gives heat resistance to enzyme including the reverse transcriptional enzyme. From this discovery, it became possible to induce reactions using the reverse transcriptional enzyme with 60°C instead of 42°C as was previously the norm. Under 60°C, the template RNAs' secondary structure gets weaker, and the area with stable secondary structure, which often exists in non-coding 5' end mRNAs, becomes possible to be transcribed in reverse in a very efficient manner.
2) Poblem: absence of actual 3' end of cDNA (i.e. 5' end of the mRNA)
Cause: inefficiency of reverse transcriptase
Solution: Cap-trapper method.
Mechanism: biotinylationof the cap-structure that is specific to mRNA of eukaryotic organisms followed by selective extraction of the biotinylated cap structure of non full-length cDNA (enzymatic degradation of ss mRNA ). The remaining full-length cDNA containing a biotinylated cap can be caught by using magnetic beads coated with streptavidin. The selected full-length cDNA can then be eluted by alkaline treatment. Synthesizing the second strand from the 1st strand cDNA (which became single stranded when separated from the beads) makes it possible to obtain the full-length cDNA selectively.
Fig: Cap Trapper method
