Biomedical Engineering Reference
In-Depth Information
1.
Perform RNA extraction, PCR amplification, and genome sequencing in different
laboratories, or at least in separate and dedicated compartments of the same labo-
ratory.
2.
Transfer reagents and samples only with aerosol-resistant pipet tips.
3.
Prepare the PCR reagent master mix in a hood dedicated for this purpose. A set of
clean gloves and dedicated lab gown should be worn in this area. Illuminate the
hood with ultraviolet before and after use.
4.
Any steps that involve the handling of cDNA, primary and secondary PCR
products (including addition of DNA templates in assembling the PCR), electro-
phoresis, and cycle sequencing should be performed in a dedicated area far away
from any PCR reagents. A separate lab gown and set of gloves should be worn in
this area.
5.
Discard all pipet tips that contacted DNA with extreme care. Use a double bag
for disposal.
6.
Include multiple negative PCR controls in each amplification to monitor for
environmental contamination.
3.2. RNA Extraction
1.
Prepare AVL lysis buffer and AW1 and AW2 wash buffers according to
manufacturer's (Qiagen) instructions ( see Note 1 ).
2.
In a biosafety level 2 (or above) containment laboratory, lyse 0.28 mL (1 vol) of
viral culture by adding 1.12 mL (4 vol) of AVL buffer, mixing and incubating at
room temperature for 10 min. Direct clinical samples, e.g., serum, nasopharyn-
geal aspirates, and stools, can also be used ( see Note 2 ).
3.
Add 1.12 mL of absolute ethanol to the mixture. Pulse-vortex for 15 s.
4.
Load the mixture to QIAamp spin column and wash the column according to the
manufacturer's instructions.
5.
Add 60
L of RNase-free water onto the membrane and incubate for 1 min at
room temperature. Centrifuge the spin column for 1 min at 6000 g .
µ
6.
Quantify a small aliquot of the extracted viral RNA yield by real-time quantita-
tive reverse-transcription (RT)-PCR ( 9 ) ( see Note 3 ).
7.
Store the extracted RNA at -80°C.
3.3. Reverse-Transcription
1.
Prewarm two thermocycler blocks with heated lid at 72 and 25°C, respectively.
2.
Mix 1
µ
L (50 pmol) random hexamer with 10
µ
L RNA in a 0.5-mL tube. Dena-
ture at 72°C for 10 min ( see Note 4 ).
3.
During this period, assemble the reaction mix in another tube on ice according to
Table 2 using SuperScript III RNase H - Reverse Transcriptase ( see Note 5 ).
4.
After denaturation, snap-cool the RNA-primer mixture on ice for 1 min. Briefly
spin the tubes. Add the reaction mix prepared in step 2 to the RNA-primer mix-
ture to make up a total reaction volume of 20
µ
L. Mix by pipetting gently up and
down.
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