Cloned amv first strand synthesis kit

Total RNA from hiPSC-RPE cells was isolated using TRIzol reagent (Sigma Aldrich, St. Louis, MO, USA), and cDNA was synthesized using the Cloned AMV First-Strand Synthesis Kit (both from Invitrogen, Carlsbad, CA, USA) [67 (link)]. Gene expression levels were analyzed by qRT-PCR (qPCR) reaction using SYBR green chemistry (Life Technologies, Carlsbad, CA, USA) and run on the ABI PRISM 7900HT platform (Applied Biosystems, Foster City, CA, USA) by a standard two-step amplification program: hot start activation (95 °C for 5 min), denaturing step (95 °C for 15 s), and annealing/extension step (60 °C for 30 s), repeated for 40 cycles. Primer sequences are listed in Supplementary Table S2. Threshold cycle (Ct) values were normalized to those of the housekeeping gene GAPDH and relative to hiPSC genes, and were expressed as 2^-ΔΔCt (log scale).

MDA-MB-231 cells were infected with oncolytic adenoviruses at MOI-100, and RNA was isolated 32 h post infection as above. cDNA was prepared using the Cloned AMV First-Strand Synthesis Kit (Invitrogen) or FireScript RT cDNA Synthesis Kit (Solis BioDyne) according to the manufacturer’s recommendations. qRT-PCR was performed on a Roche LightCycler 480 system using 5× HOT FIREPol EvaGreen qPCR Mix Plus (no ROX). Human FOXP1, MET, and PTGS2 primers were purchased as custom oligonucleotides from Invitrogen (Table S3). Hs_GAPDH_2_SG QuantiTect Primer Assay (QIAGEN, Hilden, Germany) was used for GAPDH. All reactions were done in duplicate. GAPDH-normalized FOXP1, MET, or PTGS2 gene expression was calculated using the ΔCt method; knockdown compared to irrelevant control virus-infected cells was calculated using the ΔΔCt method.

Total RNA of 5–10 eggs or larvae was extracted using TRIzol (Invitrogen) after which the RNA was purified and DNA digested on column with the RNeasy kit (Qiagen). The quality of the RNA preparation was confirmed spectrophotometrically. One microgram of total RNA was used for cDNA synthesis. First strand cDNA was made using the Cloned AMV First Strand Synthesis kit (Invitrogen). Each qRT-PCR mixture (25 μl) contained 2.5 ng of cDNA, and the real-time detection and analyses were done using SYBR green dye chemistry with the qPCR kit for SYBR Green I (Eurogentec) and a CFX96 thermocycler (Biorad). Thermal cycling conditions used were 50°C for 2 min, 95°C for 10 min, then 50 cycles of 95°C for 15 s, 60°C for 30s, 72°C for 30s. This was followed by dissociation analysis of a ramp from 65 to 95°C with a read every 0.5°C. Relative quantification for each mRNA was done using the Livak-method [30 (link)]. The values obtained for each mRNA were normalized by RPL7 mRNA amount. Total RNA for each treatment was isolated twice (biological replication) and each sample was measured by qRT-PCR twice (technical replication). Comparisons between treatments (untreated, sterile injury and septic injury) were performed within one brood.

Total RNA was isolated from LV tissue of sham, post-MI, hypo-, and hyperthyroid mice using mirVana PARIS kit for miRNA (Ambion, Foster City, CA, USA) or TriPure for mRNA (Roche, Basel, Switzerland) and treated with DNaseI (Qiagen, Venlo, The Netherlands) to remove remnants of genomic DNA, followed by reverse transcription reaction with either Cloned AMV First Strand synthesis kit (Invitrogen, Carlsbad, CA, USA) or with miRCURY LNA Universal cDNA synthesis kit II (Exiqon, Vedbæk, Denmark). Expression levels of atrial natriuretic factor (Anf), MHCα (Myh6), MHCβ (Myh7), and Dio3 mRNA were determined by qPCR using specific primers (Table S1 in Supplementary Material) and standard cycle parameters on an Applied Biosystems model 7500 (Applied Biosystems, Foster City, CA, USA) with hypoxanthine-guanine-phosphoribosyl-transferase (Hprt) as correction factor. Expression levels of miR-214-3p were analyzed using miRCURY LNA miRNA primers and normalized against U6 snRNA (Exiqon, Vedbæk, Denmark).

380 ng of RNA with 1 µM final primer concentration were employed for each RT reaction using Invitrogen’s Cloned AMV First Strand synthesis kit. Strand-specific primers; for (+) strand the reverse primers corresponding to the TSS area (+17 for mouse and +11 for human) and for (−) strand forward primers corresponding to the end of the gene (+3948 for mouse and +4791 for human, sequences in Figure S3, in File S1). Standard real-time PCR amplification of the was employed with the indicated primers and their companions, shown in Figure S2 (File S1), and the resulting Ct values were converted to relative ng and normalized to the starting concentration of chromosomal RNA.

RNA was collected as described under ‘Sample collection for transcriptional analysis’. The quality of RNA preparation was confirmed spectro-photometrically and on gel. One microgram of total RNA was used for cDNA synthesis. First strand cDNA was made using the Cloned AMV First Strand Synthesis kit (Invitrogen). Each qRT-PCR mixture (25 µl) contained 2.5 ng of cDNA, and the real-time detection and analyses were done based on SYBR green dye chemistry using the qPCR kit for SYBR Green I (Eurogentec, Seraing, Belgium) and a CFX96 thermocycler (Bio-rad, Hercules, CA, USA). Thermal cycling conditions used were 50°C for 2 min, 95°C for 10 min, then 50 cycles of 95°C for 15 s, 60°C for 30 s, 72°C for 30 s; this was followed by dissociation analysis of a ramp from 65°C to 95°C with a read every 0.5°C. Relative quantification for each mRNA was done using the Livak method (Livak and Schmittgen, 2001 (link)). The values obtained for each mRNA were normalized by RPL13a mRNA amount for Tribolium (Primers as in Lord et al., 2010 (link)). Total RNA for each treatment was isolated two times (biological replication) and each sample was measured by qRT-PCR twice (technical replication). The primers used for qPCR are in Supplementary file 3.

Total RNA was extracted from cells (RNeasy Mini Kit, Qiagen), converted into cDNA by PCR (Cloned AMV First-strand Synthesis Kit, Invitrogen; primers shown in Online Table 1), analyzed using SYBR dye (SYBR Green PCR Master Mix, Applied Biosystems).