Eurogold trifast reagent

Total RNA was extracted from RAW264.7 cells using EuroGOLD Trifast reagent (Euroclone) and quantified spectrophotometrically using a Nanodrop^®ND1000 (Thermo-Scientific). Total RNA was transcribed to cDNA using M-MLV Reverse Transcriptase (Invitrogen, Carlsbad, CA, United States). cDNA was amplified by PCR using GoTaq^®G2 DNA Polymerase (Promega, Madison, WI, United States) in the presence of the following primers (Sigma-Aldrich): C3aR forward: 5′-CCTTCTCCTTGGCTCACCT-3′; C3aR reverse: 5′-AAATACGGGCACACACATCA-3′; GAPDH forward: 5′-GCCATCAACGACCCCTTCATTG-3′; GAPDH reverse: 5′-TCTGTCATGAGGTTGGCTTTCAG-3′. The amplified samples were loaded in equal volumes onto 1% agarose gel in Tris-Acetate-EDTA (TAE) buffer, and then quantified using a Kodak Digital Science^TM Image Station 440CF system. C3aR mRNA levels were normalized to GAPDH levels.

For the RNA extraction, cells were seeded on p60 dishes at a density of 2 × 10⁶ cells/cm² in supplemented DMEM medium and incubated with iPA. Total RNA was then isolated using EuroGold Trifast reagent (EuroClone, Pavia, Italy) according to the manufacturer’s instructions. The obtained RNA samples, resuspended in 20 μL of sterile water, were measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Monza, Italy) at a wavelength of 260 nm. The RNA was then reverse transcribed into cDNA using superScript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA) starting from 1 µg of highly purified RNA. Quantitative RT-PCR was performed using gene-specific primers and a SYBR Green I (Promega Italia s.r.l, Milano, Italy) fluorescent dye. The sequences of gene-specific primers are shown in Table 2.Table 2

List of primers used.

Gene	Forward oligo	Reverse oligo
RIP1	5’- GGC ACC GCT AAG AAG AAT GG- 3’	5’- ATC GCC CAG AGT ACT ACA GC- 3’
RIP3	5’- ATA CAA CTG CTC TGG GGT GC -3’	5’- TCT TGC GAA CCT ACT GGT GG -3’
MLKL	5’- AGG ACC AAG GAA AGA GGA GC- 3’	5’-TGT CCT TTG CTG TTA GAC -3’
HMGB1	5’- CAA GTA AAT GGA AGT GGG AGG C-3’	5’- AAC CCC ACA GCA CTG TAA CT-3’
PUMA	5’- AAT GAG CCA AAC GTG ACC AC- 3’	5’- GCA GAG CAC AGG ATT CAC AG -3’
β2-MICROGLOBULIN	5’-CCT GAA TTG CTA TGT GTC TGG G-3’	5’-ACA CGG CAG GCA TAC TCA TC -3’

Total RNA was extracted using EuroGOLD™Trifast reagent (Euroclone, Devon, UK) and cDNA was synthesized using iScript™cDNA Synthesis Kit (Bio-Rad Laboratories). RT-qPCR was performed using iTaq™SYBR Green Supermix (Bio-Rad Laboratories). mRNA levels of BNIP3, VEGF-A, IL-1β, IL-18, TNF-α, IL-6, IL-10, and TGF-β were determined by MiniOPTICON™ System (Bio-Rad Laboratories) and analyzed on an iQ5™ Optical System Software (Bio-Rad Laboratories). Relative quantification was done by using the 2^-ΔΔCT method (35 (link)) and β-actin as housekeeping gene. Primers were validated as previously described (36 (link)).

Total RNA was isolated from cells 24 hours after transfection, using the EuroGold TriFast reagent (Euroclone). Random hexamers and the ImProm-II Reverse Transcriptase (Promega) were used to perform first-strand cDNA synthesis, starting from 500 ng of total RNA, according to the manufacturer's instructions. Of a total of 20 μL of the RT reaction, 1 μL was used as template for amplifications, using primers annealing to the flanking α-globin/FN1 exonic sequences (α2–3 and Bra2 primers; Fig 2). RT-PCRs were performed under standard conditions using the GoTaq DNA Polymerase (Promega) on a Mastercycler EPgradient (Eppendorf, Hamburg, Germany).
Competitive-fluorescent RT-PCRs were performed on RNA from transfected cells, using the same oligonucleotide pair adopted for splicing assays, with the reverse Bra2 primer labelled with 6-FAM. Amplified fragments were separated on an ABI-3500DX sequencer and quantitated by the GeneMapper v4.0 software (Applied Biosystems). The sum of all fluorescence peak areas in a single run was set equal to 100%, and the relative quantity of each transcript expressed as a fraction of the total.

Total RNA extraction procedures were performed using EuroGold Trifast reagent (EuroClone, Pavia, Italy; product code: EMR507100) in accordance with the manufacturer's instructions. Before extraction, cells were seeded on p60 dishes at a density of 8 × 10⁵ cells·cm⁻² in supplemented DMEM medium. The obtained RNA samples were measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific). Five hundred nanogram of purified RNA were reverse transcribed into cDNA using SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA). Quantitative real‐time PCR analysis was performed using gene‐specific primers and a SYBR Green I (Promega Italia s.r.l) fluorescent dye. Quantitative real‐time PCR reactions were run in triplicate using the CFX Opus 96 Real‐Time PCR System (Bio‐Rad Laboratories GmbH, Munich, Germany) with the cycling conditions: initial denaturation step at 95 °C for 5 min, followed by 44 cycles (95 °C for 15 s, 60 °C for 1 min). Data were analyzed using cfx manager, version 3.0 (Bio‐Rad Laboratories GmbH) and a relative quantification of gene expression was determined using the ∆∆Ct method. β‐2‐microglobulin mRNA was used as an endogenous control. All primer sequences are reported in Table 1.

Total RNA from NALM‐6 and LAL‐B cells was isolated using EuroGOLD TriFast reagent (Euroclone, IT) according to the manufacturer’s instructions. The first‐strand cDNA was synthesized with random primers and M‐MLV reverse transcriptase (Life Technologies, MA). The cDNA was used for quantitative real‐time PCR (qRT–PCR) experiments carried out in a 7500 Fast Real‐Time PCR System (Applied Biosystems, CA). ΔΔCt values were normalized with those obtained from the amplification of the endogenous β‐actin gene. The following human‐specific primers were employed in RT–PCR amplifications:
Nectin 1 forward 5’- GGATGACAAGGTCCTGGTGG- 3’
Nectin 1 reverse 5’- ACTGCACGTTGAGAGTGAGG- 3’
β - actin forward 5’ - GACAGGATGCAGAAGGAGATTACT - 3’
β - actin reverse 5’ - TGATCCACATCTGCTGGAAGGT - 3’