Amv rna pcr kit 3

Total RNA was extracted from the hypothalamus of the sacrificed rodents using TRIzol reagent (D9108A, TaKaRa, Osaka, Japan). The concentration and the purity of total RNA were examined by the A260/A280 ratio using an ultraviolet spectrophotometer (Eppendorf, Hamburg, Germany). The integrity of total RNA was also checked using agarose gel electrophoresis. According to the instructions of TaKaRa RNA PCR Kit (AMV) 3.0, all RNA samples were reverse-transcribed using the AMV reverse transcriptase (2621, TaKaRa, Osaka, Japan) and an oligodeoxythymine (oligo(dT)₁₈) (3806, TaKaRa, Osaka, Japan). All cDNAs obtained were stored at −80 °C.

RT-PCR was performed as previously described [23 (link)]. Briefly, total RNA was isolated from HUMSCs and HUMSC–SC co-cultures using Trizol reagent (Invitrogen, New York, USA), according to the manufacturer’s instructions. RT-PCR was performed using the RNA PCR Kit (AMV) 3.0 (TaKaRa, Da lian, China, Japan). PCR conditions were 2 minutes at 94°C, then 35 cycles of 94°C for 30 seconds, 50 to 65°C for 30 seconds, 72°C for 30 seconds, and a final extension for 10 minutes at 72°C. PCR products were resolved by 1.2% (w/v) agarose gel electrophoresis, visualized by ethidium bromide staining, and photographed under ultraviolet light. The human housekeeping gene β-actin was used as a normalization control. The sequences of human-specific primer sets used for PCR were as follows: VASA (NM_024415.2, 191 bp), forward 5’-AAG AGG TAG TTT CCG AGG TTG C-3’and reverse 5’-CTT TGT AAC CAC CTC GTT CAC T-3’; DAZL (NM_001351, 487 bp), forward 5’-ATC ATC CTC CTC CAC CAC AG-3’ and reverse 5’-GAT TTA AGC ATT GCC CGA CT-3’; STELLA (NM_199286, 315 bp), forward 5’-CTC CAC AAA TGC TCA CCG AA-3’ and reverse 5’-GCT CCT TGT TTG TTG GTC TTC T-3’; and β-actin (NM_001101, 396 bp), forward 5’-CAC ACT GTG CCC ATC TAC GA-3’ and reverse 5’-TAC AGG TCT TTG CGG ATG TC-3’.