Rna to cdna kit

RIP was performed using a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, 17-700) according to the manufacturer’s instructions. HUVECs in 15-cm dishes were collected and resuspended in cold RIP lysis buffer, and then stored at –80 °C overnight. Magnetic beads were preincubated with 1 µg of PTBP1 antibody (Invitrogen, 32-4800) or with 1 µg of control IgG (Millipore, 03-241). Next, the frozen homogenates were thawed quickly and centrifuged at 18,000 × g for 10 min, and 10 µl of the supernatant was stored as input. Then, for each RIP reaction, 100 µl of supernatant was incubated overnight with the magnetic bead-antibody complex at 4 °C. On the second day, the RNA/protein immunocomplex was extensively washed with RIP Wash Buffer (provided in the kit). The cross-linking was reversed by incubation with proteinase K. The immunoprecipitated RNA was purified through phenol:chloroform:isoamyl alcohol (125:24:1) isolation. The purified immunoprecipitated RNA was reverse-transcribed into cDNA using an RNA-to-cDNA Kit (Takara, RR036A). qRT-PCR was performed using this cDNA as a template to quantify the ARRB1 mRNA; the following primers were used: ARRB1 (forward: 5’-TACAGTCGTTCCCACCGG-3’ and reverse: 5’-GACGCACAGAATTCCGCT-3’) and GAPDH (forward: 5’-GTCTCCTCTGACTTCAACAGCG-3’ and reverse: 5’- ACCACCCTGTTGCTGTAGCCAA-3’).

Total RNA was isolated from purified parasites using an RNA purification kit (Qiagen). cDNA was synthesized from 1 μg of total RNA using an RNA-to-cDNA kit (Takara). Pbqsox expression was determined by qRT-PCR using primers Pbqsox1 and Pbqsox2 (Table S1). qRT-PCR reactions consisted of 2 μl cDNA, 10 μl SYBR Green fast master mix (ThermoFisher), 0.5 μl each of the forward and reverse primers and 7 μl RNase-free water. Analysis was conducted using a 7500 Fast PCR System (ThermoFisher) with the following conditions: initial denaturation at 95°C for 20 s, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. The expression of hsp70 (PBANKA_081890) was used as the internal reference with primers Hsp70F and Hsp70R (Table S1). Relative quantification of pbqsox expression was assessed using the ΔΔC_t method (Livak and Schmittgen, 2001 (link)).

Samples from pancreatic cancer and adjacent non-tumor tissues and from different experiment cell groups were used to extract the total RNA by Trizol reagent (Invitrogen) Using 500 ng RNA to reverse transcript to cDNA using the RNA-to-cDNA kit (TaKaRa, USA). And then quantitative real-time PCR (qRT-PCR) reactions were carried out using the Power SYBR Green PCR Master Mix (TaKaRa, USA). The RT products were detected by PCR reaction which denatured for 30 seconds at 95°C, 5seconds for 95°C, 30s for 60°C by forty cycles in all. All the data were described using the comparative quantitative threshold cycle (ΔΔCt) method.

RNA was isolated from kidney tissue using the Qiagen RNeasy kit as described by the manufacturer. cDNA was synthesized using Takara Bio RNA to cDNA kit as described by the manufacturer. 100ng of cDNA was used per reaction with BioRad SYBR green mix and primers as listed in Supplementary Table S2. Ultrapure DNase-/RNase-free water was used as a no template control (NTC). Expression of each gene was corrected for expression of β-actin.

P. berghei RNA was extracted from P. berghei-infected mouse blood 4 days after infection, and cDNA was synthesized using an RNA-to-cDNA kit (Takara). The pbph gene was amplified with primers pbph-F and pbph-R (Additional file 1, Table S1) using P. berghei cDNA as the template. The PCR fragment was cloned into the expression vector pET30a (+) at the NdeI and HindIII sites and transformed in Escherichia coli BL-21. Protein expression was induced at 20 °C for 12 h in the presence of isopropyl-β-D-thiogalactoside (Promega) at a final concentration of 1 mM and 1 % anhydrous D-glucose. Bacterial cells were lysed on ice by sonication for 15 cycles (20 s pulses with 30 s intervals between each cycle) and the resultant suspension passed through a 0.22-μm filter. The filtered supernatant was loaded onto a Ni-NTA His•Bind Superflow column (Millipore) and purified according to the manufacturer’s protocol. Purified protein was dialyzed overnight in phosphate buffered saline (PBS, pH 7.2) containing 0.2 mM PMSF at 4 °C and an aliquot was analyzed on a 12 % SDS-PAGE gel. Protein samples were quantified using a Bradford assay.