Tri reagent

Total RNA was isolated from cell lines, primary cells, normal canine tissues (lung, liver, heart, pancreas and kidney), and primary lymphoma tissues from client-owned dogs presenting with clinical lymphoma using Tri-reagent (Molecular Research Center, Inc.) (Table 2 and S1 File).
All cells and frozen tissue samples (40-80mg) were homogenized in 1 ml Tri-reagent. RNA was isolated from the aqueous phase by isopropanol precipitation. RNA concentration was determined by absorbance at 260 nm. The mRNA expression from canine survivin, CXCR4, TERT, and beta actin (cSurvivin, cCXCR4, cTERT) was measured by quantitative reverse transcriptase PCR (Q-RT-PCR). The cDNA from 1ug of RNA was synthesized using an iScript cDNA synthesis kit (Bio-Rad, Catalog # 1708891). One tenth of the RT reaction volume was used for Q-RT-PCR assays using SSO-Advanced Universal syber green supermix (Bio-Rad) with 0.5uM forward and reverse primers in a volume of 20ul (Table 3). Thermocycling conditions were: 3 min at 95°C, then 40 cycles of 95⁰ C for 30 sec and 57°C for 30 sec, on a Bio-Rad iCycler iQ Multicolor Real-Time PCR Detection System. The mRNA expression was analyzed by the ΔCt method using beta-actin as the comparator. Primer specificity was validated by sequencing the PCR product (Eurofins MWG Operon).

Total cellular RNA and DNA were extracted using TRI Reagent (Molecular Research Center, Inc., Cincinnati, OH) as per manufacturer’s instructions, with the following modifications: polyacryl carrier (Molecular Research Center, Inc., Cincinnati, OH) was added to TRI Reagent prior to lysis, RNA was resuspended in RNase free-water, DNA was extracted using back extraction buffer (4M guanidine thiocyanate, 50mM sodium citrate, 1M Tris), polyacryl carrier was added to the aqueous phase containing the DNA, and DNA was resuspended in QIAGEN buffer EB.