Superscript 2 reverse transcriptase enzyme

For RNA isolation SKMEL28, 451LU and BLM melanoma cell aggregates were used. Total RNA was isolated by using RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions, including DNase I treatment to remove contaminating genomic DNA (Invitrogen). Purified RNA samples were used for reverse transcription (RT) reaction containing Oligo d(pT)18 mRNA primers (New England Biolabs, Frankfurt, Germany) and Superscript II reverse transcriptase enzyme (Invitrogen) according to the supplier's protocol. The human TGFbeta BMP signaling pathway RT2 Profiler PCR array (Qiagen) comprising a total of 84 genes was performed according to the manufacturer's instructions. For this project, only the data for BMP-2 (CT-values) were included.

RNA Extractions from cells or tissues were performed as per the instructions of the Nucleospin RNA II kit (Macherey Nagel). 1μg RNA was reverse transcribed to cDNA as per the instructions accompanying the Superscript II Reverse Transcriptase enzyme (Invitrogen). The resulting cDNA was used for qualitative or quantitative PCR analysis. For qualitative PCR analysis, the cDNA was purified using a PCR purification kit (Invitrogen). Of the purified cDNA, 1–2μl was subjected to PCR with Taq polymerase (Invitrogen) or Pfu polymerase (Promega). In each case, PCR conditions were followed according to the kit instructions. PCR products were then analysed by agarose gel electrophoresis (1–2%) The primers used for this purpose are listed in Table 1.
Quantitative Real-Time PCR was performed on the 7900HT Real-Time System (Applied Biosystems). The relative expression of target genes was analysed by the SDS 7900 Software. Primers used for this purpose are listed in Table 1. The dissociation curves and primer efficiencies were monitored using the SDS 7900 Software.

BMDMs were seeded at 1 x 10⁶ cells per well into 6-well plates in RPMI-1640,allowed to adhere for 4h, and then conditioned, as described above, with 4T1- or HC11-conditioned DMEM for 24 h. The cells were lysed and total RNA was obtained using the Qiagen RNeasy Mini kit (Toronto, ON, Canada), following the manufacturer’s protocols. RNA concentration was assessed using a NanoDrop spectrophotometer and 500 ng of total RNA was reverse transcribed using SuperScript II Reverse Transcriptase enzyme (Invitrogen, Burlington, ON, Canada) following manufacturer’s protocols. Real-time PCR was performed in a 10 μl reaction, with 1 μl of 1/10 diluted cDNA, using the FastStart SYBR Green Master kit (Roche, Mississauga, ON, Canada), following manufacturer’s instructions. Melting curves were performed to verify the production of specific amplicons. The primer sequences against the coding sequence of each chemokine (Integrated DNA Technologies, Coralville, IA, USA) are presented in Table 1. Changes in gene expression were calculated by normalizing the change in expression of each gene of interest to that of housekeeping gene GAPDH using the delta-Ct method. Gene expression of each chemokine by conditioned BMDMs was then normalized to that of non-conditioned BMDMs, yielding fold change-over-control values.