Iscript rt

Total RNA from heart biopsies was extracted using the RNeasy Fibrous Tissue Mini kit (Qiagen GmbH, Hilden, Germany) according to the indications provided by the company. A small aliquot of the total RNA obtained (1 μL) was subjected to qualitative and quantitative control by using the microdrop (Thermo Fisher Scientific, Waltham, Massachusetts). We qualitatively and quantitatively assessed the individual samples using dedicated software. Total RNA was reverse-transcribed into cDNA by using iScript RT (Bio-Rad, Hercules, California). SYBR Green gene expression assays were performed in triplicate according to the manufacturer's instructions using the iQ SYBR Green Supermix (Bio-Rad, Hercules, California) and the iQ5 Multicolour Real-Time PCR Detection System (Bio-Rad, Hercules, California). The pairs of primers used are listed in Table S1.

CD4⁺ T-cells and MDDC total RNA was isolated using RNeasy Plus Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacture instruction. Then, 250 ng and 500 ng RNA respectively from CD4⁺ T-cells and DCs were reverse transcribed into cDNA using iScript RT (Bio-Rad laboratories, Hercules, CA, USA).

To determine the tissue distribution of GRP, bombesin and GRPR mRNA in X. tropicalis (n = 3 of each sex), RT-PCR analysis was performed. Total RNA was extracted from various tissues (brain, spinal cord, heart, lung, stomach and skin), using illustra RNAspin Mini RNA Isolation Kit (GE Healthcare). First-strand cDNA was synthesized from 200 ng of total RNA in a 20-μL reaction volume using oligo-dT primers and iScript RT (Bio-Rad Laboratories). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control. Primer pairs are shown in Supplementary Table S2. The resultant PCR amplicons were electrophoresed on 2% agarose gels. RT-PCR studies were repeated four times using independently extracted RNA samples from different animals. Consistent results were obtained from each run.

Total RNA from liver and small intestine biopsies was extracted using the RNeasy Plus Mini Kit (Qiagen GmbH, Hilden, DE) according to the indications provided by the company. A small aliquot of total RNA obtained (3 μl) was subjected to qualitative and quantitative control by using the microdrop (Thermo Fischer Scientific, Waltham, MA). The qualitative and quantitative assessment of the individual samples was determined using a dedicated software. The total RNA was reverse transcribed into cDNA by using iScript RT (Bio-Rad Laboratories, Hercules, CA)^{82 (link)}. SYBR Green gene expression assays were performed according to the manufacturer’s instruction using the iQ™ SYBR® Green Supermix (Bio-Rad Laboratories, Hercules, CA) and the CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA).
Primer sequences are reported in Supplementary Table S6.
mRNA expression levels were normalized to β2-microglobulin and quantification of relative gene expression, presented as percentage of the relevant baseline, was calculated using the 2-∆CT (comparative threshold) method.

DNA and RNA were isolated from normal human lungs as described previously. Total RNA was reverse transcribed to yield cDNA using iScript RT (Biorad) and random primers as per the manufacturer's instructions (Biorad). PCR was carried out using primers that were designed flanking the editing sites to amplify at least a 400 bp product. Primers were designed such that they could be used on both genomic DNA (gDNA), and spliced cDNA. We also tagged primers with M13 primers at the end so that it could be amenable to Sanger sequencing using M13 primers alone. Amplified PCR products were purified using PCR purification kit (Qiagen) and subjected to Sanger sequencing using M13 primers at both ends using standard protocol at Moffitt Genomics facility. The potential edited sites were considered validated if the cDNA sequence at the edited site contained two peaks, (both a reference peak and an edited peak) but the corresponding gDNA contained only one reference peak. Sites were considered to be un-validated if it contained only a single reference peak in both cDNA and gDNA traces.