SCARPET oligonucleotides can have nonspecific cleavages that increase background signal. (A) SCARPET underestimates m6A stoichiometries when the concentration of the target RNA is low. SCARPET results are shown for a site in a 42-nt long synthetic RNA standard comprising a 1:1 mixture of methylated and nonmethylated RNA oligonucleotide standards, with amounts ranging from 1.25 to 40 fmol added to 1 µg 2×-poly(A) HepG2 mRNA. At low concentrations of the RNA standard, SCARPET underestimated m6A stoichiometries. Only at the highest levels (40 fmol of each RNA standard) did the estimated m6A (46.3%) get close to the actual stoichiometry of 50%. (B) SCARPET results using a SCARPET oligonucleotide specific for ΦX174 A2768, an arbitrary site, using 1 µg total RNA from HEK293T cells as the input. In principle, no nucleotide should be detected since the ΦX174 RNA is not present in HEK293T RNA. However, a SCARPET oligonucleotide-dependent A spot was detected, demonstrating nonspecific cleavage of cellular RNA. (C,D) SCARPET result for another arbitrary site in ΦX174 (A580). A SCARPET oligonucleotide-dependent A spot was detected. A weak A spot in the absence of the SCARPET oligonucleotide was detected, which likely reflects some ligation of the hairpin oligonucleotide to spurious 5′ ends with weak complementarity to the hairpin oligonucleotide. One microgram of 2×-poly(A) HEK293T mRNA and 1 ng of purified MYC HEK293T mRNA were used as input. (C). The A spot completely disappeared when using purified MYC mRNA as the input which suggests that using purified mRNA can lower the background, and therefore provide more accurate quantification in SCARPET. The RT-qPCR-based fold enrichment of purified MYC mRNA relative to GAPDH mRNA in the pull-down with MYC mRNA targeting capture oligonucleotides compared to MALAT1 lncRNA which was used as a nonspecific control (D). Note: Both ΦX174 A2768 and A580 sites are present in the common GGACU consensus site for m6A. 32P-labeled m6A and A nucleosides were run as TLC standards in A–C.
