RNA Detection

2. Add loading buffer to each of the reactions, mix thoroughly
   and centrifuge for several seconds.


3. Carefully load DL2000 DNA marker and samples into differ-
   ent lanes of the gel.


4. Run agarose gels at 150 V for 40 min, until the sample line
   reaches approximately 75–80% of the way down the gel.


5. Visualize the gels at UV light (Fig.2).

3.3 Two-
Dimensional HCR for
an Oligo Target on
Solid Surface (See
Fig.3)

1. Dilute target probe (Target oligo) to 10, 1, 0.1, 0.01, and
   0.001 nM respectively in hybridization buffer (seeNote 6).


2. Add 100μL of above solutions to individual capture wells.


3. Seal the wells with tinfoil sealing film and incubate at 46C for
   1h(seeNote 7).


4. Tear the film, then quickly decant the wells and wash with
   300 μL of wash buffer for three times, with quick decanting
   in between (seeNote 8).


5. Centrifuge the plate upside-down for 1 min at 600
   gafter the
   final wash and decant.


6. Mix 120μL of hybridization buffer, 15μL of probe X1*, and
   15 μL of probe X2 (seeNotes 9and 10 ).


7. Add 100μL of above mixture to the capture plate, seal and
   incubate at room temperature for 2 h.


8. Repeat wash steps (steps 4and 5 ).


9. Mix 120μL of hybridization buffer, 15μL of probe A1, and
   15 μL of probe A2.


10. Add 100μL of above mixture to the capture plate, seal and
    incubate at room temperature for 2 h.


11. Repeat wash steps (steps 4and 5 ).


12. Add 150μLof4
    SYBR Green I in 4
    SSC buffer to the
    reaction wells, and then incubate at room temperature for
    15 min in the dark place.


13. Quantify the resulting fluorescence with a plate reader.

3.4 Direct RNA
Detection in Blood
Sample (See Fig.4,
Note 11)

1. Thaw the blood sample withP. falciparumat 4C before use
   (seeNote 3).


2. Lyse 10μL of thawed blood sample with 50μL of lysis mixture,
   85 μL of water, and 2μL of 50 mg/mL proteinase K at 60C
   for 1 h with vigorous shaking.


3. Mix the lysate with 1.5μL of respective CEs and LEs probes
   (seeNote 12).


4. Add 100μL of the above mixture to the capture plate, then seal
   the wells with tin foil and incubate at 58C overnight without
   shaking (seeNote 13).

Hybridization Chain Reaction for Direct RNA Detection 193