PAGE 3 OF 7 Nucleic Acids Research, 2010, Vol. 38, No. 7 e98 Figure 1. Eﬀect of various hybridization buﬀers on the sensitivity of LED protocol in detecting miR-21 and miR-16. Seven diﬀerent hybridization buﬀers (A–G) based on a probe concentration of 0.2 nM were used as indicated and detailed in supplementary document (Supplementary Table S1). Varying amounts of total RNA (3, 5 and 10 mg) were used to detect mature miR-21 and miR-16 (arrowheads) for each probe concentration, and the corresponding photo-luminescence was recorded over varying lengths (1, 3 and 5 min) of time. The upper bands may correspond to the precursor and primary transcripts of the miRNAs. more sensitive than others in detecting miR-21 while ULTRAhyb, DIG Easy Hyb, buﬀer C, Church–Gilbert (9) and modiﬁed Church–Gilbert buﬀer (10) yield readily detectable, linear, concentration-dependent signals for both miR-21 and miR-16. The Church–Gilbert buﬀers are also useful in detecting miRNAs due to their linear detection range, and provide a cost- and time-advantage over other tested buﬀers since they can be readily prepared from a few reagents. Since ULTRAhyb, and buﬀers B/C contain formamide that reduces the melting temperature for probe–RNA hybridization and hence may be a factor in their perfor- mance, we also tested modiﬁed Church–Gilbert protocol with 50% formamide at a reduced temperature (37 C). The formamide containing protocol did not yield a better signal than ULTRAhyb (Supplementary Figure S3) and did not appear to have any notable improvement over the protocol without formamide. The evaluations of eight diﬀerent buﬀers allowed us to select a set of three buﬀers [ULTRAhyb, Buﬀer A and modiﬁed (10) Church–Gilbert] that could be further optimized and analyzed. ULTRAhyb and Buﬀer A were selected due to their high sensitivity, while the modiﬁed Church–Gilbert protocol was chosen due to its ease of availability and reduced cost. We note that buﬀer C and DIG easy Hyb, is likely as useful as buﬀer A, yet we selected buﬀer A for further tests because buﬀer A appeared to be more sensi- tive in detecting the low abundance, putative primary miR-16 transcript (Figure 1, upper most band). Optimization of hybridization-temperature conditions Based on the three selected buﬀers, we next studied the eﬀect of hybridization temperature on sensitivity (Supplementary Figure S4). Consistent with the manufacturer-recommended conditions for DNA/RNA probes ( 40–60 C), our results show that there is not a large diﬀerence between sensitivities at 37 and 60 C for