Myeloid Leukemia

PRV-1 Overexpression in PV and ET 269
3.3. Reverse Transcription and Testing to Verify the Presence of cDNA
3.3.1. Reverse Transcriptase Reaction
1. Mix the total RNA that has been subjected to DNase treatment (8 µL for each
sample) with 1 µL of dNTPs and 1 µL of Oligo(dT) 12–18.
2. Incubate the tube at 65°C for 5 min, then place the sample on ice for 2 min.
3. Add 9 µL of reaction mixture composed by 2 µL of 10X reverse transcriptase
buffer, 4 µL of 25 mM MgCl 2, 2 µL of 0.1 M DTT, and 1 µL RNaseOUT to each
RNA/primer mixture, mix gently, and incubate at 42°C for 2 min.
4. Then, add 1 µL (50 U) of SuperScript II RT to each tube except for the no RT
control, mix, and incubate at 42°C for 50 min.
5. Stop the reaction by heating at 70°C for 15 min.
6. Add 1 µL of RNase H into each tube and incubate for 20 min at 37°C.
7. Store at –20°C.
3.3.2. Test to Verify the Presence of cDNA
Reverse transcribed RNA (cDNA) is amplified with primers for the housekeeping
gene β-actin to control for the presence of cDNA in each sample (see
Note 2).
1. Amplify 3 µL of cDNA in a reaction mix containing specific primers for the βactin
gene (1 µmol/L of each primer), 1 unit of Taq DNA polymerase, 200 µmol/
L of dNTPs, 2.5 µL of 10X Reaction Buffer A, 1.5 mM MgCl 2, and water to a
final volume of 25 µL. The β-actin-specific oligonucleotide primers (forward 5�-
TAC ATG GGT GGG GTG TTG AA-3�; reverse 5�-AAG AGA GGC ATC CTC
ACC CT-3�) will amplify a 234-bp fragment.
2. The PCR conditions are as follows: one cycle of 3 min at 95°C, followed by 36
cycles at 95°C for 40 s, 55°C for 40 s, 72°C for 40 s, and a final cycle of 3 min at
72°C.
3. Separate the PCR products on a 2% agarose gel in 1X TBE buffer.
4. After staining with ethidium bromide, visualize the PCR products under UV light
(Fig. 1).
3.4. PRV-1 Amplification and Detection
1. Amplify 3 µL of cDNA in a reaction mix containing specific primers for PRV-1
(1 µmol/L of each primer), 1 U of Taq DNA polymerase, 200 µmol/L of dNTPs,
3 µL of 10X Reaction Buffer A, 1.5 mM MgCl 2, and water to a final volume of 30
µL. The PRV-1-specific primers (sense 5�-CAG TTT GGG ACA GTT CAG C-
3�; antisense 5�-AAA GCG GGA GGG AGT TAA C-3�) will amplify a 286-bp fragment.
2. The PCR conditions are as follows: one cycle of 5 min at 95°C, followed by 31 cycles