Abi 7500 qpcr system

RNA extraction was performed using the RNA Clean and Concentrator kit (Zymo Research, Irvine, CA) and eluted into 15 μl H₂O. RNA from tissue was then converted into cDNA using SuperScript VILO Master Mix (Life Technologies, Grand Island, NY). The RNA was assumed to be converted 100% to cDNA. PCR reactions were run in 30 μl using target specific, Taqman hydrolysis probes for the gene of interest and were normalized to Gapdh (Ref 4351317, Applied Biosystems/Life Technologies, Carlsbad, CA). Normalized gene expression was determined by differences in the cycle thresholds (Ct) between genes of interest and Gapdh (ΔCt) on a ABI 7500 qPCR System (Applied Biosystems). The viral vector-injected SN (“ipsilateral”) was examined for transcript expression of the transgene human, wildtype Snca (Applied Biosystems assay ID Hs01103386_m1). Robust expression was required on the ipsilateral side for inclusion in this experiment; no transgene expression was detected on the contralateral side. The SN was also examined for the following transcripts (Applied Biosystems Assay ID# following): rat, wildtype Snca (Rn00569821_m1), Bdnf (Rn02531967_s1), Th (Rn00562500_m1) and Trk2 (Rn01441749_m1).

Total RNA was isolated, and complementary DNA (cDNA) was synthesized with a Primer Script TM RT kit (Takara, Dalian, China). Targeted GAPDH (housekeeping gene), Bcl-2, Bax, Fas, caspase-3, caspase-8, and p53 primers were prepared using the Primer Premier 5.0 software (PREMIER Biosoft, Palo Alto, CA, USA) and generated by Shanghai Biotechnology Co. Ltd (Shanghai, PR China). qPCR was performed using an ABI 7500 qPCR system (Applied Biosystems, Foster City, CA, USA) and qPCR Detection Kit (Takara) as per kit instructions under the following conditions: denaturation (94 °C, 3 min), denaturation (40 cycles, 94 °C, 30 s), annealing (52–55 °C, 30 s), and elongation (72 °C, 30 s). Each experiment was repeated three times and performed in duplicate in 96-well plates. Relative gene levels were computed using the 2^−△△Ct formula, and alterations in gene levels were evaluated via the Student’s t-test (P < 0.05 was set as the significance threshold).

Total RNAs were extracted from cells using TRIzol reagent (Invitrogen) and reversed transcribed into cDNAs using PrimeScript RT Master Mix (Takara, Dalian, China). qPCR was then performed to detect the expression levels of miR-3934 in different samples using SYBR premix Ex Taq (Takara) on an ABI 7500 qPCR System (Applied Biosystems Life Technologies, Foster City, USA). Relative expression levels of miRNA-3934 were determined using the 2^−ΔΔCt method and were normalized to U6. The PCR protocol entailed 95 °C for 30 s followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd (Shanghai, China). The primers used for real-time PCR were as follows: RAGE forward primer: 5′-GTGTCCTTCCCAACGGCTC-3′, reverse primer: 5′-ATTGCCTGGCACCGGAAAA-3′; GAPDH forward primer: 5′-TGTGGGCATCAATGGATTTGG-3′, reverse primer: 5′-ACACCATGTATTCCGGGTCAAT-3′; miR-3934-5p forward primer: 5′-GCCAGCTCCTACATCTCAGC-3′, reverse primer: 5′-AGCCTGACTTGCTAGTGGATTAT-3′; and U6 forward primer: 5′-CTCGCTTCGGCAGCACA-3′, reverse primer: 5′-AACGCTTCACGAATTTGCGT-3′.