Improm 2 protocol

Total RNA was isolated from cultured cell lines using a RNAzol B reagent (Biotecx Laboratories, Houston, TX) according to the manufacturer’s instructions, and then cDNA was prepared from 2 μg of the total RNA with random hexamer primers according to the cDNA synthesis ImProm-II protocol (Promega). The specific oligonucleotide primer pairs for human CXCL16 were forward 5′-GAG CTC ACT CGT CCC AAT GAA-3′, reverse 5′-TCA GGC CCA ACT GCC AGA-3′; for human beta-actin, forward 5′-GCC AAC CGC GAG AAG ATG A-3′, reverse 5′-CAT CAC GAT GCC AGT GGT A-3′; for interleukin-8, forward 5′-CCA GGA AGA AAC CAC CGG-A-3′, reverse 5′-GAA ATC AGG GCT GCC AAG-3′; for human angiogenin, forward 5′-CCT GGG CGT TTT GTT GTT GG-3′, reverse, 5′-TGT GGC TCG GTA CTG GCA TG-3′; and for human G9a, forward 5′-CCG GCG CAA GGC CAA GAA GA-3′, reverse 5′-CGG TGG GCC ACA CGG AAG TC-3′. The amplification program consisted of one cycle of an initial incubation at 61°C for 20 min, followed by 50 cycles of denaturation at 95°C for 10 sec, annealing at 55–57°C for 10 sec, and then extension at 72°C for 10 sec. The amount of indicated mRNA was normalized by that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA and is presented in arbitrary units, with 1 U corresponding to the value in cells treated with a vehicle control.

Total RNA was extracted with the TRIzol reagent from 1 × 10⁶ B16F10 cells or 100 mg of tissue sample following manufacturer instructions (Invitrogen). One microgram of total RNA was used for DNase treatment and subsequent reverse transcription using the ImProm-II protocol (Promega). For semiquantitative PCR, target genes were amplified using 100 ng of DNA and Taq platinum DNA polymerase (Invitrogen) following the manufacturer protocol. Samples were loaded in a 2.0% agarose gel stained with SYBR Safe (Invitrogen). For quantitative real-time PCR, we used 10–50 ng of complementary DNA (cDNA) and platinum SYBR Green qPCR SuperMix UDG with ROX reference dye (Invitrogen); results were analyzed using the ABI Prism 7000 sequence detection system. Transcripts were quantified relative to the housekeeping gene beta-actin using the Ct method (Livak and Schmittgen, 2001 (link)). The oligonucleotide primers used in the PCR analyses are listed in Tables S1 and S2.

RT–PCR was used to verify LPA specific receptor expression. Total RNA was isolated from CHON-001 cell line using RNAzol B reagent according to the manufacturer's instructions. cDNA was prepared from 2 µg of total RNA with random hexamer primers according to the cDNA synthesis ImProm-II protocol (Promega). The specific oligonucleotide primer pairs and the expected sizes of the PCR products are showed in Table 2. The PCR products were electrophoresed on a 1.8% agarose gel at 100 V and visualized using ethidium bromide.