Each reaction mixture contained 10 uL SYBRA(r) Premix Ex TapTM II, 1 uL of each primer (10 uM), 0.5 uL of ROX Reference Dye II, 1 uL of cDNA and 6.5 uL of RNase-free
water in a final volume of 20 uL.
To synthesize cDNA, 1 [micro]g of RNA and 1 [micro]L each of oligodT (Invitrogen, USA) and RNase-free
water were added.
Each 20 [micro]L reaction solution contained 10 [micro]L 2xmiRNA Reaction Buffer Mix, 2 [micro]L 0.1% BSA, 2 [micro]L miRNA PrimeScript RT Enzyme Mix, 1 [micro]L total RNA, and 5 [micro]L RNase-Free
[H.sub.2]O; qRT-PCR was carried out for Initial activation of 95[degrees] C for 10 s, followed by 40 cycles of 95[degrees]C for 5 s, 60[degrees]C for 20 s.
The precipitate was then dissolved with a proper amount of RNase-free
H2O, providing purified RNA.
Briefly, 5 u l of undiluted first RT-PCR product was added to a reaction mixture consisting of 3 u l of 25mM MgCl2, 2 u l of 10mM dNTPs, 0.5 u l of Taq Polymerase (5U/ul), 5 u l of 10X Taq buffer, 1 u l of each type specific primers P, P and P and an upstream primer Con2 and RNase-free
water to final volume of 50 l, and subjected to nested multiplex PCR amplification by following the PCR cycles as reported by Falcone et al.
Subsequently, a nested PCR was carried out by using the following: 3 [micro]l of RT-PCRproduct, master-mix: 14 [micro] l, forward and reverse primers: 1 [micro]l each, and RNase-free
water: 6 [micro]l to make a final volume of 25 [micro]l.
Reaction was performed in RNase-free
water and incubated for 10 min at 37[degrees]C with shaker at low speed (300 rpm).
The following materials were purchased from the manufacturers indicated: RNeasy Kit and RNase-Free
DNase from Qiagen (Hilden, Germany); High-Capacity cDNA Reverse Transcription Kit and Power SYBR Green PCR Master Mix from Applied Biosystems (Foster City, CA); GenElute PCR clean-up kit, 3-3'-diaminobenzidine, heparinase I and III blend from Flavobacterium heparinum, and heparan sulfate from Sigma-Aldrich (St.
The cDNA was synthesized in a 0.2 mL RNAse-free
microtube, starting with 5.0 [micro]L of viral RNA.
The real-time PCR reaction system was a 10 [micro]L reaction mixture containing 0.5 [micro]L cDNA, 4.1 [micro]L RNase-free
water, 5 [micro]L 2x SYBR Green Master Mix, and 1.8 [micro]L of each primer (10 mM).
Materials required were the following: buffer RAV1 2x 35 mL, buffer Raw 2x 30 mL, buffer RAV3 (concentrated) 2x 125 mL, buffer RE 2x 5 mL, carrier DNA lyophilized 2x 1mg, Ribo Virus columns 100, collection tubes (2) 300, ethanol 96-100%, biological cabinet, microcentrifuge tubes, sterile RNAse-free
pipette tips with aerosol barrier, disposable gloves (powder-less), microcentrifuge (with rotor for 2mL tubes), and real time qPCR machine.
After the microcentrifuge tube was dried, the RNA pellet was resuspended in 20 [micro]L of ultrapure RNase-free