Modeling

Summary

We examined the efficiency of circularization of mRNA in E. coli. The mRNA synthesized by transcription of induced plasmid may be considered that there are mainly 3 shape patterns in the process of circularization (fig.1). In this experiment, we carried out reverse transcription of RNA of Escherichia coli, and calculated the abundance ratio of each shape pattern by detecting specific sequences by using MPN-PCR (Most Probable Number-PCR).

By examining the abundance ratio of sequence A, B or C, D , and B / A shows the probability that the reaction doesn’t start (step Ⅰ). Also, we can calculate the probability that RNA will cyclize (step Ⅱ,Ⅲ) by C, D. By examining these, we investigated the rate-limiting step in the process of cyclization or probability to cyclization of the RNA.

Experiment

We extracted total RNA from E. coli and then carried out the reverse transcription. This time we used the two types of primer; oligo dt primer and random primer. And we carried out reverse transcription with them. We serially diluted obtained cDNA, and calculated abundance rate by using MPN-PCR.

Case 1: Determination of the sequence A and B

We carried out reverse transcription with the oligo dt primer which complementary to the poly A sequence at the 3 'end of the mRNA (fig.2).

Oligo dt primer anneals specifically to the 3 'end of the mRNA, so the ratio of reverse transcription of A and B are the same. Thus, B/A calculated by the MPN-PCR shows the abundance ratio of step Ⅰ, that is, the probability that no reaction started.

Case 2: Determination of the sequence C and D

Since there’s no poly A sequence on the mRNA after starting cyclization, it’s impossible to use the oligo dt primer. Therefore, we use the random primer. However, some problems arise when we determine them with random primer.