Question

Design an oligonucleotide-based probe for purifying all the mature mRNA from eukaryotic cell lysates.

Answer 1

For Purifying all mature mRNA from eukaryotic cell lysate, we can be use the method called tandem RNA ISOLATION PROCEDURE (TRIP ). In this method streptavidin beads are tagged with 3' biotinylated 2' O methylated antisense RNA oligonucleotide.

Tandem RNA isolation procedure (TRIP) that enables the purification of endogenously formed mRNA-protein complexes from cellular extracts. The two-step protocol involves the isolation of Polyadenylated mRNAs with antisense Oligo(dT) beads and subsequent capture of an mRNA of interest with 3'-biotinylated 2'-O-methylated antisense RNA oligonucleotides, which can then be isolated with streptavidin beads.

ANTI SENSE OLIGONUCLEOTIDE

1. Secondary analysis of mRNA analysis using one of online tool called as VIENNA RNA Website with suitable option such as MFE (minimum free energy ) partition function and avoid isolated base pair option. All eukaryotic cell mRNA have poly AA Tail at 3' end of mRNA thus oligonucleotide with poly T tail could be design over streptavidin bead which will capture all mRNA with poly A tail.
2. For preferentially specific mRNA , the probe oligonucleotide should have 21-24 nucleotide long sequence which do not have secondary structure and binds with 3' untranslated region (3UTR) of mRNA. There should not be tandem repeats to avoid potential formation of hairpin or self annealing as well as must have 50% G/C ratio.
3. Another way is manually design 2' Methoxy Modification RNA Nucleotide with biotin moiety at 3' and is fully complementary to target mRNA. 2' methoxy allow protection from cellular RNase present in cell lysate and duplex melting Temperature increases allow stringent washing leading to high purified mRNA.