Figure 1.
Schematic illustration of microRNA capture by 3′ adapter ligation.
The 19 nt, enzymatically pre-adenlyated adapter is ligated to the 3′ OH of microRNA using T4 RNA ligase 2. The reaction is run at 25°C for 4 hours in the absence of ATP. In order to characterize capture efficiency, the microRNA is end labeled with Cy3. The 3′ end of the adapter is blocked by –ddC, a fluorophore, or other moiety to prevent the formation of concatemers and circularized products.
Figure 2.
MicroRNA capture was performed with 4 different ligases using the vendor recommended protocols to compare capture efficiency across 20 different microRNA.
The ligation products were analyzed by 15% denaturing urea-PAGE. Capture efficiency was determined by performing a Cy3 scan and comparing the intensities of the ∼40 nt captured microRNA band versus the ∼20 nt free microRNA band. T4 RNA Ligase 2 truncated (T4 Rnl2 T) had high average capture efficiency and low bias but many randomly sized background products. The point mutant enzymes T4 RNA Ligase 2 truncated K227Q (T4 Rnl2 TK) and T4 RNA Ligase 2 truncated KQ (T4 Rnl2 TKQ) had decreased side product formation but also lower average capture efficiency and higher bias. Thermostable 5′ App DNA/RNA Ligase (Mth Rnl), which was performed at 65°C instead of 25°C, had similar average capture efficiency and bias but with distinct ligation efficiency pattern.
Figure 3.
Comparison of ligation efficiency as a function of PEG percentage for miR-31, miR-155, and miR-4803 in idealized buffer (open markers) and total RNA spiking conditions (filled markers).
miR-155 and miR-4803 display similar behavior with respect to PEG percentage while miR-31 behaves distinctly. Different behavior is also seen between idealized buffer conditions and total RNA spiking conditions, illustrating the importance of optimization methodology in extrapolating assay performance. Optimizations performed using a single microRNA species in idealized buffer may not extrapolate to other microRNA under actual assay conditions.
Figure 4.
The adapter concentration and T4 Rnl2 TK amount were changed simultaneously to see their joint effect on the capture efficiency of miR-31.
The experiment was performed under idealized buffer conditions and total RNA spiking conditions which lead to distinct conditions for optimum capture efficiency. Under spiking conditions, greater amounts of adapter and enzyme are necessary to obtain high capture efficiency.
Figure 5.
The ligation reaction was incubated at 25°C for 30 min to 18 hours to investigate the effect of time on the capture efficiency of miR-31 in idealized buffer conditions and total RNA spiking conditions.
In idealized buffer conditions, the ligation reaches completion in <2 hours. Whereas in spiking conditions, the ligation does not reach full completion until >8 hours.
Figure 6.
Comparison of incubation temperature on capture efficiency across the 20 microRNA panel.
The ligation reaction was incubated for 18°C, 4 hours at 25°C or 4 hours at 37°C under total RNA spiking conditions.
Figure 7.
Comparison of capture efficiency across the 20 microRNA panel spiked into 500 ng of total RNA using 4 different adapter designs.
The rA and dA adapters have identical sequences based on modified modban design except the 5′ base is either RNA or DNA as indicated. The SR1 and SR1-S adapters are taken from Zhuang et al (39). T4 Rnl2 TK shows no preference for DNA or RNA at the ligation site. However, overall capture efficiency and bias were significantly worse for the SR1 and SR1-S adapters.
Figure 8.
The optimized capture efficiency for the 20 microRNA panel in idealized buffer conditions and total RNA spiking conditions.
The data is presented as representative gel images and as graphs based on image analysis of 3 sets of independent experiments. High capture efficiency and low bias are obtained across the panel both in idealized buffer conditions and total RNA spiking conditions.