Goscript reverse transcriptase system

Dissections, RNA extraction, and cDNA synthesis were performed as described in Calkins et al. [11 (link)]. In brief, mosquitoes were anesthetized on ice before being dissected in mosquito Ringer solution (consisting of 150 mM NaCl, 3.4 mM KCl, 1.7 mM CaCl₂, 1.8 mM NaHCO₃, 1 mM MgCl₂, 5 mM Glucose, 25 mM HEPES; pH 7.1). Tissues were isolated, transferred to 1.5-ml micro-centrifuge tubes (Thermo Fisher Scientific, Waltham, MA, USA), and preserved in TRIzol^® reagent at −80 °C until utilized in RNA isolation. Total RNA was isolated using the method of Chomczynski and Sacchi [17 (link)] and quantified using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). cDNA libraries were synthesized using 4 µg of total RNA and the GoScript^TM Reverse Transcriptase system with random primers (Promega, Madison, WI, USA), following manufacturer’s protocols. cDNA libraries were stored at −20 °C until needed for qPCR.

Extracted RNA was reverse-transcribed into cDNA using the Goscript
^TM Reverse Transcriptase System (Promega, Madison, USA) according to the manufacturer’s instructions. Semi-quantitative PCR was performed using a PCR kit (Tiangen, Beijing, China) on a thermo-cycler (Bio-Rad, Hercules, USA). PCR reactions were performed in a total volume of 25 μL containing 1 μL cDNA. Primers of the TRPV4 channel were designed for semi-quantitative PCR. All specific primers for the TRPV4 channel were designed based on Genbank mouse sequences. Specific primer sequences are as follows: TRPV4 channel, sense: 5′-TACGACCTGCTGCTTCTCAA-3′, antisense: 5′-TCCTCATCTGTCACCTCACG-3′; and GAPDH, sense: 5′-GGTGAAGGTCGGTGTGAACG-3′, antisense: 5′-CTCGCTCCTGGAAGATGGTG-3′. The cDNA product was PCR amplified using specific primers for the TRPV4 channel. The PCR annealing temperature for each primer pair was optimized using a Master Cycler Gradient Thermocycler (Eppendorf, Hamburg, Germany). PCR reaction consisted of 30 s denaturation at 94°C, 30 s for annealing at 58–64°C, 30 s for elongation at 72°C, and 30 amplification cycles. PCR products (15 μL) were separated by electrophoresis on a 1% agarose gel in Tris-borate/EDTA buffer. The quantification of semi-quantitative PCR was performed by using the optical density (OD) method.

For the analysis of uhrf1 expression, wildtype and hmx1 knockout embryos at 24 hpf and 5 dpf were euthanized. For whole body transcript analysis at 24 hpf and 5 dpf, 60 embryos obtained from different breeding were pooled together. For eye transcript analysis, both eyes were isolated from 20 embryos at 5 dpf and pooled together. All experiments were repeated three times.
First-strand cDNA synthesis was performed using the AffinityScript^™ Multiple Temperature Reverse Transcriptase kit (Agilent, Basel, Switzerland) according the manufacturer’s protocol. cDNA was generated (GoScript Reverse Transcriptase System; Promega) and Real time PCR (FastStart SYBR Green Master Roche) was performed using uhrf1-F 5’-TCCAGGAGTCCAAGAGAGGAA-3’ and uhrf1-R 5’-TCTGCTGAACACAGTTCGGG-3’; dnmt1-F 5’-TTACTTTGGGCAAGAGGAGAGC-3’, dnmt1-R 5’-GACACCACACCGTTGTCTCT-3’; hmx1-F 5’-CGAAACCTCCAGGAG TCCAAG-3’, hmx1-R 5’-CGGGTCTTTTTCTTTCGGGC-3’.
Gene expression change was determined using the 2^–ΔΔCt method; relative values were normalized with β-actin.

Microarray data were verified by quantitative real time PCR. Briefly, First-strand cDNA was synthesised using random hexamer primers and the GoScript Reverse Transcriptase System (Promega), and qPCR was performed using the SYBR green PCR protocol (Applied Biosystems) on the Chromo4 real-time PCR system (Bio-Rad). Primer sequences are presented in S5 Table. Each experiment included ‘no template’ controls, was run in duplicate and had an 18S RNA control. Each independent experiment was repeated three times, and the results were analysed by independent-samples t-test.

RNA for gene expression analysis from EndoC-βH1 was extracted using TRIzol reagent (Invitrogen) and synthesized into complementary DNA using the GoScript Reverse Transcriptase System (Promega). RNA for gene expression analysis from primary human pseudoislets was extracted using the PicoPure RNA isolation kit (Life Technologies), and cDNA was synthesized using the Maxima first strand cDNA synthesis kit (Thermo Fisher Scientific), according to the manufacturer’s instructions. Quantitative qPCR (qPCR) was performed using TaqMan real-time PCR assays on a 7900HT Fast Real-Time PCR System (all Applied Biosystems, Supplementary Table 4). Ct-values were analyzed using the ΔΔCt method, and target genes were normalized to the combined average of the housekeeping PPIA, GAPDH and TBP. CALCOCO2 expression in EndoC-βH1 and primary islets was extracted from previously published and analyzed RNA-seq data^{71 (link),72 (link)}.

Cells were harvested, and total RNA was isolated using the PureLink™ RNA Mini Kit (Thermo Fisher Scientific, Cat. No. 12183020) as per the manufacturer’s instructions. First-strand cDNA synthesis of 250–500 ng total RNA was done using GoScript Reverse Transcriptase System (Promega, Catalog No. A5004). Relative mRNA expression of target genes was checked using SYBR® Green Real-Time qPCR Master Mix (Thermo Fisher Scientific, Cat. No. 4309155) in QuantStudio™ 7 Flex Real-Time PCR System, 96-well block (Applied Biosystems™, Cat. No. 4485701). The primer sequences used are listed in Table 1.

Total RNA was extracted using a ReliaPrep™ RNA Cell Miniprep System (Promega) and quantified by a Nanodrop 2000 (Thermo, Wilmington, DE, USA). For RT-PCR, 0.5 μg of total RNA from each sample was used for cDNA synthesis using the GoScript™ Reverse Transcriptase system (Promega). Real-time PCR was performed on a real-time PCR system (7500 Fast Machine, Applied Biosystems, Thermo) using GoTaq^® qPCR Master Mix (Promega). The comparative Ct (∆∆Ct) method was used to calculate the gene expression levels after normalization, according to the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The data show the relative values to the mRNA levels when cultured on control tissue culture plastic. The primers and their sequences are shown in Table 1.