e98 Nucleic Acids Research, 2010, Vol. 38, No. 7 ULTRAhyb using LNA-based probes. However, back- ground eﬀects clearly improve upon increasing tempera- ture (37–60 C). We also found that the modiﬁed Church–Gilbert protocol performs best at 60 C and its sensitivity is comparable to that of ULTRAhyb and Buﬀer A for detecting miR-21. Although the sensitivity of buﬀer A was comparable between the optimal temper- ature conditions of 37 C and 50 C, the performance of buﬀer A manifested greater variability than both ULTRAhyb and the modiﬁed Church–Gilbert protocol (Figure 1 and Supplementary Figure S4). Speciﬁcally, in contrast to previous experiments using buﬀer A, the band intensities during the temperature-optimization experi- ments for buﬀer A are weaker and are linear with RNA concentration. Such inconsistencies (additional data not shown) of buﬀer A and the increased sensitivity of ULTRAhyb over the modiﬁed Church–Gilbert protocol in detecting miR-16 led us to choose ULTRAhyb (37 C) as the preferred buﬀer solution for further evaluation of LED. Although we selected the ULTRAhyb buﬀer for further tests due to its overall performance, in our experi- ence the modiﬁed Church–Gilbert protocol generally gen- erates less background than ULTRAhyb, and is clearly an eﬀective buﬀer for detecting abundant miRNAs. Eﬀect of membranes on LED performance Since we relied on our experience in using positively charged Nylon membrane (Roche) for previous experi- ments, we sought to systematically test whether diﬀerent membranes could signiﬁcantly aﬀect LED performance. Among the four diﬀerent membranes tested (Figure 2), we found that the selected positively charged membrane (Roche) and the neutral Nylon membrane (Hybond-NX, GE) performed best (Figure 2A and B). Surprisingly, another widely used positively charged membrane (Bright star plus, Ambion) yielded relatively weak signals with moderate background levels (Figure 2C) in Figure 2. Evaluation of four diﬀerent nylon membranes for LED protocol. Duration of photo-exposure (1, 3 and 5 min) and amount of total RNA (3 and 6 mg) are indicated. Among the tested membranes (A–D), positively charged and neutral nylon membranes purchased from Roche (A) and GE Healthcare (B), yielded the strongest signals. PAGE 4 OF 7 LED protocol. The amphoteric (Figure 2C) membrane performed poorly, and did not yield any signals for either miR-21 or miR-16. The results conﬁrmed that the selected membrane (Roche) for LED is a reasonable choice, and also highlighted the inﬂuence of diﬀerent membranes and membrane sources on northern blots used for small RNA detection. Comparison of LED to other methods To better understand the contribution of LNA probes, EDC cross-linking and DIG labeling to the LED protocol, we systematically tested their eﬀects on the per- formance of northern blots in detecting miR-21 and miR-16. Speciﬁcally, one by one, we substituted LNA probes, EDC cross linking and DIG labeling by DNA probes, UV cross-linking and 32P labeling, respectively (Figure 3). As expected, the LNA-based protocols are con- siderably more sensitive than DNA-based northern blots (Figure 3: A versus B, C versus D, E versus F and G versus H). In comparison to 32P labeling, the use of DIG also signiﬁcantly improves performance of the blot (Figure 3: A versus C and E versus G). Although the use of EDC cross-linking consistently improves the sensitivity of the method (Figure 3: A versus E and C versus G), it increases the background in DIG labeling-based method. Figure 3. Systematic evaluation of the contribution of probe type, RNA–membrane cross-linking and probe labeling to LED perfor- mance. LNA probes, EDC-based cross-linking and DIG labeling in LED protocol were systematically substituted by DNA probes, UV-based cross-linking and 32P labeling to generate the following eight combinations for detecting miR-21 and miR-16: LNA-EDC-DIG (A) corresponds to LED protocol (A), DNA-EDC-DIG (B), LNA-EDC-32P (C), DNA-EDC-32P (D), LNA-UV-DIG (E), DNA-UV-DIG (F), LNA-UV-32P (G) and DNA-UV-32P (H). Phosphor image screens were used for all protocols to eliminate any bias due to the imaging system (‘Materials and Methods’ section). Duration of photo-exposure (1 min and 24 h) and amount of total RNA (3 and 6 mg) are indicated.