PROTOCOL

1. Ethylenediaminetetraacetic Acid (EDTA) Whole Blood Processing

Note: Blood can be processed immediately after collection of can be stored at 4 °C

for no more than 24 hr.

1. Working in a biosafety cabinet, allow the EDTA whole blood sample to reach room
temperature.
2. For each sample, label enough cryovials with the sample identification (ID), storage
material (plasma), and date.
3. Centrifuge the samples for 10 min at 1,000 x g. Do not use brakes to stop centrifuge.
This will yield three layers (from top to bottom): plasma, leucocytes (buffy coat) - a
very thin layer - and erythrocytes, including platelets.
4. Carefully aspirate the supernatant (plasma) and aliquot 500 ml into each cryovial.
Take care not to disrupt the cell layer (buffy coat) or transfer any cells.
5. Store at -80 °C until needed for RNA extraction or proceed to RNA extraction
immediately.

2. RNA Extraction

1. Prepare an extraction worksheet with the IDs of the samples to be extracted

including positive and negative plasma controls.
2. For each sample to be extracted, label a 1.5 ml sterile microcentrifuge tube with the
sample ID, extraction date and “RNA”. Also label an assembled column and
collection tube as well as a 2 ml microcentrifuge tube containing working lysis
solution with corresponding numbers from the extraction worksheet.
3. Working in the Bio-Safety Cabinet, add 200 µl sample to the corresponding 2 ml
microcentrifuge tube of working lysis solution.
4. Vortex well and incubate for 10 min at room temperature.
5. After 10 min, centrifuge the tube briefly.
6. Add 800 ml of absolute ethanol to each of the tubes.
7. Mix by pulse vortexing and briefly centrifuge.
8. Transfer 600 µl of this solution to the corresponding column/collection tube
assembly. Centrifuge at 6,000 x g for 1 min.
9. Transfer column to a new collection tube and discard the old collection tube
containing the filtrate. Repeat the above step 2.8 (above) twice more.
10. Add 500 µl wash buffer AW1 to each column and centrifuge at 6,000 x g for 1 min.
11. Discard the filtrate and collection tube and transfer the column to a new collection
tube.
12. Add 500 µl was buffer AW2 and centrifuge at 20,000 x g for 3 min. Repeat step 2.11.
13. Centrifuge in a new collection tube at 20,000 x g for an additional 2 min.
14. Discard filtrate and place column in 1.5 ml microcentrifuge tube.
15. Add 60 µl Buffer AVE (RNase free water) to the middle of the column ensuring that
you do not dispense the liquid on the side of the column.
16. Incubate at room temperature for 1 min.
17. Centrifuge at 6,000 x g for 2 min.
18. Discard the column and cap the 1.5 ml microcentrifuge tubes.
19. The samples are now ready for reverse transcription.
20. If testing is to be performed immediately, store at 4 °C for up to 6 hr. However, if
testing is to be delayed then place at -80 °C immediately. NB: do not freeze/thaw the
samples more than 3x.

3. Reagent Preparation for Reverse Transcription

1. Before starting, calculate the volumes of each of the reagents required for the
number of samples being processed including, the positive and negative plasma
controls. Also add a reagent control.
2. Using the calculated volumes from step 3.1 (above), prepare the
deoxyribonucleotide triphosphate (dNTP)-primer mix in a clean, sterile 200 µl PCR
tube followed by briefly pulse vortexing. Each sample should have 0.5 µl of the
reverse primer RT21 and 0.5 µl of the dNTP, see Table 2.
3. Aliquot 1.0 µl of the dNTP-primer mix to 200 µl PCR tubes.
4. Prepare reverse transcriptase (RT) enzyme mix by adding 1 µl of the 10x reverse
transcription buffer, 1 µl of 0.1M DTT and 2 µl of 25mM MgCl 2 to a sterile tube
followed by vortexing and briefly centrifuging, see Table 3.
5. Add 0.5 µl each of the enzymes RNAseOUT and Superscript III reverse transcriptase
to the enzyme mix tube then tap the tube gently to mix.
6. Keep the tubes with the dNTP-primer mixes and enzyme mix on a cold block and
move to the RNA station.

4. Reverse Transcription

1. Add 6 µl of the RNA sample to the dNTP-primer mix tube followed by briefly
vortexing to mix.
2. After the addition of the RNA, move to the PCR room with both dNTP/primer/RNA
mix and RT Enzyme mix tubes on a cold block or ice.
3. Briefly centrifuge the dNTP/primer/RNA mix tubes (from step 4.2) and place them
into a thermocycler.
4. Heat at 65 °C for 5 min to denature the RNA.
5. Rapidly cool to 4 °C, hold for 2 min.
6. Pause the thermocycler while still at 4 °C; take out the tubes.
7. Quickly add 5 µl of the enzyme mix while keeping the tubes on a cold block.
8. Mix gently by tapping the tube then briefly centrifuge the tubes and return to the
thermocycler.
9. Hold the tubes at 50 °C for 60 min to reverse transcribe the RNA followed by enzyme
denaturation at 85 °C for 5 min to stop the reverse transcription.
10. Cool to 37 °C. As soon the temperature gets to 37 °C, pause and take the tube out of
the thermocycler.
11. Quickly add 0.5 µl of RNAse H to the tubes and return to the thermocycler.
12. Hold at 37 °C for 20 min and then cool to 4 °C.
13. The complementary DNA (cDNA) can be used immediately or can be stored at -20
°C or colder until needed. However, the long term storage of cDNA should be at -80
°C.

5. Reagent Preparation for PCR

1. Before starting, calculate the volumes of each of the reagents required for the
number of samples being processed and the controls. In addition to the three
controls (Positive, Negative, and Reagent), you can also add a PCR control (HIV
DNA). The first and second round PCR mixes can be prepared simultaneously and
the second master mix stored at -20 °C until needed. Mixes can be stored for
approximately 8 hr.
2. Add 18.4 µl water, 2.5 µl 10x buffer, 1.0 µl MgCl2, 0.5 µl dNTPs, and 0.25 µl of each
of the primers as shown on Table 4 and vortex.
3. Add 0.1 µl of Platinum Taq polymerase (5U/µl) and gently mix the tube by tapping it.
4. Aliquot 23 μl of the master mix to 200 μl PCR tubes.
5. With the master mix tubes on a cold block or ice move to the PCR room.

6. Nested PCR

1. Add 2 µl of the cDNA to 23 µl of the 1st round PCR master mix.

2. Close the tubes, put the samples in the thermocycler and use the following PCR
cycling conditions: 94 °C for 2 min, 30 cycles of 95 °C for 30 sec, 58 °C for 20 sec,
and 72 °C for 2 min, followed by a final extention at 72 °C for 10 min as shown
on Figure 1.
 Figure 1. Nested PCR cycling conditions. Click here to view larger image.

3. Continue to the 2nd round PCR stage or store the 1st round PCR products at -20 °C
or colder until required at a later stage.
4. For 2nd round PCR, add 2 µl of the 1st round PCR product to 23 µl of the 2nd round
PCR master mix and use the same PCR program on Figure 1.

7. Gel Electrophoresis

1. Gel preparation
1. Add a 0.5 g of agarose tablet to a 250 ml glass flask and add 50 ml of 1x TBE buffer
to the flask.
2. Heat in microwave to boiling; swirl frequently (approximately every 30 sec) until
completely solubilized. Use a silicone grip or silicone oven glove to grasp the hot
flask. The agarose solution can boil out of the flask very easily so closely monitor this
process.
3. Cool at room temperature for approximately 10 min.
4. Pour agarose into a gel casting tray containing appropriate size comb; gel is ready to
use in approximately 20-30 min.
5. Place gel in electrophoresis chamber and run as recommended by the manufacturer.
2. Gel electrophoresis and visualization.
1. Vortex Novel Juice for 10 sec prior to use.
2. Dilute 1 µl of Novel Juice with 5 µl of DNA sample and mix.
3. Dilute 3 µl of Novel Juice with 3 µl of molecular weight marker and mix.
4. Load the mixes from sections 7.2.2 and 7.2.3 (above) and run the gel at 100 V and
400 mA for 40 min to evaluate the PCR amplification.
5. Positive amplification can be visualized under UV light as 1,315 bp fragment, Figure
2.
 Figure 2. Gel confirmation of PCR amplification using 1% agarose gel
electrophoresis and a 200 bp ladder. Click here to view larger image.
6. There should be no amplification in the Negative and reagent controls, thus
indicating absence of contamination.

8. PCR Product Cleanup

1. In preparation for the sequencing reaction, the positive second round PCR products
are cleaned up using the PureLink PCR purification kit.
2. Add 80 μl of working Binding buffer High-Cutoff (B3) to 20 µl of PCR product and
pipette mix.
3. Add the sample mixed with the binding buffer to a spin column in a collection tube.
4. Centrifuge the column at 10,000 x g for 1 min. Transfer the column into a new
collection tube.
5. Wash the column with 650 µl of Wash Buffer with ethanol.
6. Centrifuge the column at 10,000 x g for 1 min. Transfer the column into a new
collection tube.
7. Centrifuge the column at maximum speed for 2-3 min to remove any residual wash
buffer.
8. Place the spin column in a clean 1.7 ml elution tube supplied with the kit.
9. Add 40 µl of elution buffer to the center of the column and incubate the column at
room temperature for 1 min.
10. Centrifuge the column at maximum speed for 2 min (>10,000 x g).
11. The elution tube contains your purified PCR product ready for sequencing. Discard
the column.
12. Determine the concentration and quality of the DNA using a Nanodrop.
13. If no in-house sequencing facilities are available, the purified PCR products can be
sent to a commercial sequencing lab at this stage.