Quantitect sybr green rt pcr assay kit

qPCR was performed using the Quantitect SYBR-green RT-PCR assay kit (Qiagen) and an ABI 7900 thermocycler. Data was normalized relative to β-actin, whose splicing is not affected by this level of SF3a1 inhibition [26 (link)]. Primer sequences used for qPCR are listed in S21 Table. qPCR was performed in triplicate and analyzed with Graphpad Prism 5 using t-tests to determine statistical differences (p<0.05).

Semi-confluent cells plated in 60 mm tissue culture dishes were treated in triplicate for 6 h with forskolin (0, 0.1, 0.3, 1, or 3 μM) plus rikkunshito stock solution (0 or 3%). Total RNA was extracted by using the QIAshredder (Qiagen, Hilden, Germany) and the RNeasy Mini Kit (Qiagen). First strand cDNA was prepared by using oligo-dT primers and SuperScript III reverse transcriptase (Invitrogen, Life Technologies). Rat tyrosine hydroxylase (TH) and rat VIP mRNA levels were measured by using the QuantiTect SYBR Green RT-PCR assay kit (Qiagen) and a real-time monitoring fluorescent quantitative detection system (LineGene, Bioflux, Tokyo, Japan). The mRNA levels for each gene were normalized relative to those of glyceraldehyde-3-phosphate dehydrogenase (G3PDH). The primer sequences were as follows: TH, forward 5′-AGCCC AAGGG CTTCA GAAGG GC-3′, reverse 5′-CGCTC CTTGC GGGCA TCCTC GATGA G-3′; VIP, forward 5′-CGCTG GCCTG GCCTC TCTAT G-3′, reverse 5-CTGCA AGATG TCAGA GTCTG C-3′; and G3PDH, forward 5′-TGCAC CACCA ACTGC TTAGC-3′, reverse 5′-AGTGA TGGCA TGGAC TGTGG-3′.