- UV transilluminator.
- Resolving gel buffer and running buffer (1TBE): 90 mM
Tris, 89 mM boric acid, and 2.0 mM EDTA, pH 8.0.
Add about 100 mL water to a 1 L graduated cylinder. Weigh
10.89 g Tris, 5.50 g boric acid, and 0.75 g EDTA, and then
transfer to the cylinder. Add water to a volume of 900 mL. Mix
and adjust pH with HCl. Make up to 1 L with water. Store at
room temperature. - 1% Agarose with 1 ng/mL ethidium bromide (seeNote 1).
- DL2000 DNA marker: Store at 4C.
- Loading buffer.
2.2 RNA Detection 1. Tabletop centrifuge with plate adapter.
- Heat incubator.
- Fluorescence plate reader.
- Tinfoil sealing film.
- Dissolving buffer: TE.
- Stock hybridization buffer (20 SSC): 3 M NaCl, 0.3 M
sodium citrate, store at 4C. - Hybridization buffer: 5SSC (diluted from 20SSC), store at
4 C. - Wash buffer: 0.1SSC containing 0.3 g/L lithium dodecyl
sulfate, store at 4C. - Capture 96-well plate functionalized with a 22-base DNA
sequence (capture probe, Fig.4), (Diacurate), store at 4C
(seeNote 2). - Blood sample withP. falciparum, store at 20 C(seeNote 3).
- Lysis mixture (Diacurate), store at room temperature.
- 50 mg/mL Proteinase K, store at 20 C(seeNote 3).
- Hairpin sets: DNA sequences designed independently using
NUPACK 5. - 100μM oligonucleotide probes in TE: include CEs and LEs,
designed by Diacurate to specifically capture malaria RNA,
store at 20 C. Dilute all probes to 10μM with hybridization
buffer before use (seeNote 4).
l CE (Capture Extender).
CE probe contains two regions: region I on 3^0 -end has the
complementary sequence with capture probe and region II
on 5^0 -end can hybridize with target RNA. A “TTTTT”
sequence is inserted between the two regions.
l LE (Label Extender).
Hybridization Chain Reaction for Direct RNA Detection 191