Fig. 1
Optimizing the conditions for single-tube amplification (STA) of RNA. a Schematic representation of STA procedure. TSO: template-switch oligo and primer binding site for PCRIIA; i7: primer binding site for FC-121-1030; P5 and P7: flow cell binding sites for Illumina sequencing; red: ribonucleic acid bases; green: locked nucleic acid bases. b Denaturing PAGE (6%) of LOrA5 under various conditions. One pmol of LOrA5 was polyadenylated with 2.5 units of PAP in 10 μl of reaction medium at 37 °C for 30 min followed by incubating at 65 °C for 20 min with the following variations: I: RNase If 25 units at 37 °C for 30 min after the 65 °C step; A: RNase A 5 μg at 37 °C for 30 min after the 65 °C step; heat: the reaction was heated at 65 °C for 20 min before adding LOrA5 for polyadenylation. PAPB: PAP buffer; RTB: RT buffer; ATP: 2.5 mM final of ATP. Arrow: LOrA5; arrowhead: LOrA5 with trimmed rA tails by RNase If. c Schematic representation of extending a looped DNA oligo (LO) with a TSO. d Denaturing PAGE (12%) demonstrating the effects of Mg2+ and dNTP concentrations on TS activity. One pmol of LO was incubated in 1 M betaine, 1 μM TSO, 1X RT buffer, 4 mM extra of dithiothreitol, 50 units of superscript II in 10 μl of reaction medium at 42 °C for 90 min followed by incubating at 70 °C for 15 min with different final concentrations of dNTP and MgCl2. Lanes 8 and 9 served as no-LO and no-TSO controls, respectively. Arrow: LO with first extended TSO; asterisk: LO with second extended TSO. e Schematic representation of detecting a DNA oligo with a 20-mer A tail (mT7A20) without (Control) or with (PreAmp) preamplification. f Denaturing PAGE (12%) detecting decreasing numbers of mT7A20 using STA. Various numbers of mT7A20 were subject to TS reaction as described in the Methods section. A half (5 μl) of the TS reaction was directly purified with polyethylene glycol (PEG)/NaCl, and all eluents underwent 50 cycles of PCR (Control, product 3 in Fig. 1e). The other half (5 μl) was preamplified with 20 cycles of PCR before purification (PreAmp, product 5 in Fig. 1e), and one-hundredth of the eluents was amplified with 40 cycles of PCR. g Denaturing PAGE (12%) of miR-19b PCR products with decreasing cell inputs. Total RNA from 1000- to 1-cell lysates of 293FT was amplified based on the STA procedure (21 cycles). One-hundredth of the purified eluents was subject to 40 cycles of PCR. 293FT-RT: control (1000 cells) without reverse transcriptase; 293FT-PAP: control (1000 cells) without PAP; 0: no-cell control. h Nondenaturing PAGE (15%) demonstrating detection limits of STA with spike-in of small-RNA oligos. Various numbers of 27-mer RNA (RNA27) oligos were spiked into 100-cell lysates of TW1 hESC line. One-hundredth of the purified eluents from the 20-cycle STA procedure was subject to 40 cycles of PCR