Superscript 3 platinum sybr green one step qpcr kit w rox

RT-qPCR analysis was performed with SuperScript III Platinum SYBR Green One- Step qPCR Kit w/ROX (Invitrogen) and the ABI 7300 Real-Time PCR System. The 96-well RT-qPCR plate was prepared by following the manufacturer's recommendation. Five nanograms of each sample were added into each well. Each reaction was performed in triplicate in 25-μl reaction volumes, with 16S rRNA as a control. For each reaction, 200 nM primers (Supplementary Table S2) were used for RT-qPCR.

Total RNA was extracted from LCM-captured excitatory neurons and astrocytes with the NucleoSpin miRNA kit (Macherey-Nagel, 740971). Q-RT-PCR was performed with a StepOnePlus Real Time PCR System (Applied Biosystem) using the SuperScript III Platinum SYBR Green One-Step qPCR Kit w/ROX (Invitrogen, 11746). The PCR cycling condition was as follows: initial denaturation at 95°C for 5 min followed by 50 cycles of 95°C for 15 s and 60°C for 30 s and a final extension at 40°C for 1 min. Ct values were generated by StepOne Software version 2.2.2. The expression level of each gene was normalized to B2m. All primer sequences are provided in S3 Table. The PCR product was purified with NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel, 740609) and analysed by Sanger sequencing.

To detect intracellular encapsidated pgRNA, HepG2.2.15 or HepAD38 cells were lysed with 300 μl of lysis buffer (10 mM Tris-HCl [pH 7.6], 100 mM NaCl, 1 mM EDTA, and 0.1% NP-40) per well. Cell debris and nuclei were removed by centrifugation, and the supernatants were digested with 20 U/ml of micrococcal nuclease (MNase) at 37°C for 30 min. The core particles were precipitated with 35% PEG 8000 dissolved in 1.5 M NaCl on ice for 60 min, isolated by centrifugation, and dissolved in TNE buffer (10 mM Tris-HCl, pH 7.6, 150 mM NaCl, and 1 mM EDTA). Encapsidated pgRNA in core particles was extracted with TRIzol reagent, and pgRNA was quantified in a qPCR assay (SuperScript III Platinum SYBR Green one-step qPCR kit w/ROX; Invitrogen) with the 5′-GGT CCC CTA GAA GAA GAA CTC CCT-3′ (sense) and 5′-CAT TGA GAT TCC CGA GAT TGA GAT-3′ (antisense) primers using the PCR conditions of 50°C for 30-min hold (cDNA synthesis), denaturing at 95°C for 5 min, followed by 40 cycles of amplification at 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s.