Superscript 4 vilo master mix kit

Nucleic acid from samples submitted to VIDRL was extracted using the Qiagen Qiamp 96 kit and a Qiacube HD extraction robot as per the manufacturer’s instructions (Qiagen, Hilden, Germany). RNA was reverse transcribed using Bioline’s Sensifast synthesis kit (Bioline, UK) as per kit instructions. Nucleic acid from samples collected at CHW was extracted using the Qiagen Viral RNA mini kit (Qiagen, Hilden, Germany) and RNA was reverse transcribed using Life Technologies’ Superscript IV VILO master mix kit (Thermofisher Scientific, Victoria, Australia) as per manufacturers’ instructions. cDNA was stored at −20 °C until processing.

Total RNA was extracted from a pool of eight-week-old rice leaves from each plant and reverse transcribed to make cDNA using the SuperScript IV VILO Master Mix kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions.
Quantitative real-time PCR (qRT-PCR) was performed using cDNA and the PowerUP SYBR Green master mix (Thermo Fisher Scientific) with a CFX96 real-time PCR detection system (Bio-Rad, Hercules, CA, USA). Normalized expression (2(-Delta Delta C(T) method) was calculated using the Bio-Rad CFX manager software, employing the housekeeping gene OsActin1 (Os03g50885) as a reference gene. All experiments were performed in triplicate for technical repeats. The results were plotted as relative values ±SEM and graphically displayed using GraphPad Prism version 8.2.0 (GraphPad Software, Inc., San Diego, CA, USA). The qRT-PCR for each gene was repeated three times. The primer sequences used are listed in Table S2.

Total RNA was reverse transcribed using a SuperScript IV VILO MasterMix kit (Thermo Fisher Scientific). For relative expression of circANRIL junctions in EndoC cells and human islet samples by qPCR, Taqman primer and probe sets for the detection of ANRIL junctions 7–5, 10–5, 16–5, and 16–4 were designed. Together with two commercial TaqMan ANRIL expression assays ANRIL (Exon5-6), Hs04259476_m1; and ANRIL (Exon1-2), Hs01390879_m1, as well as those for CDKN2B p15INK4b, Hs00793225_m1; CDKN2A p16INK4a, Hs02902543_mH; CDKN2A ARF, Hs99999189_m1, the expression levels of target genes were quantitatively assessed in duplicate by normalizing to the level of endogenous reference (ACTB, Hs01060665_g1; and GAPDH, Hs02758991_g1) Transcript expression levels were presented as log2-transformed expression (ΔCt).

EndoC cells were collected for subcellular fractionation and total RNA isolation. Cytoplasmic and nuclear RNA were isolated with the Cytoplasmic & Nuclear RNA Purification Kit (Norgen, Belmont, CA, USA) following the manufacturer’s manual. Briefly, EndoC cells were harvested and incubated with a lysis buffer for 5 min on ice. Then, the cells were centrifuged at maximum speed for 10 min at 4 °C, the supernatant was kept for assessing the cytoplasmic RNA, and the pellet was used for nuclear RNA extraction. Next the RNA was reversely transcribed to cDNA according to the instructions of SuperScript IV VILO MasterMix kit (Thermo Fisher Scientific). The expression levels of ANRIL (Exon5-6), Hs04259476_m1; ANRIL (Exon1-2), Hs01390879_m1, circular ANRIL (7–5, 10–5, 16–4 and 16–5) in the whole cells, nuclei and cytoplasm were examined by qRT-PCR. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and Nuclear Enriched Abundant Transcript 1 (NEAT1) were detected as fractionation indicators. The primers for GAPDH RNA were 5'-CTCCTCCTGTTCGACAGTCA-3' (sense) and 5'-GTTGACTCCGACCTTCACCT-3' (antisense). The primers for NEAT1 RNA were 5'-GTGGCTGTTGGAGTCGGTAT-3' (sense) and 5'-TAACAAACCACGGTCCATGA-3' (antisense).

Total RNA was extracted and purified from pooled scrambled control and genotyped cldn7b F0 knockout larvae (n = 50 per group) at 7dpf using the RNeasy Plus Micro Kit (QIAGEN), followed by cDNA synthesis with the SuperScript IV VILO Master Mix Kit (ThermoFisher). Forward (5′-AATCCTCTCTGTTGGAGCCCT-3′) and reverse (5′-TTGACAGGTGTGAAGGGGTTG-3′) primers spanning the cldn7b exon 2-exon 3 junction were designed using Primer BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). Each qRT-PCR reaction (10 µl/well) was assembled by adding 2 µl 0.5 ng/µl cDNA, 0.5 µl 10 µM forward and reverse primer mix, and 5 µl 2X iTaq Universal SYBR Green Supermix (Bio-Rad Laboratories). Experiments were run in three technical replicates on a CFX384 Touch Real-Time PCR Detection System (Bio-Rad Laboratories). Relative gene expression fold change was calculated using the Livak method (2^−ΔΔCt)^{58 (link)}. GAPDH was the housekeeping gene chosen for expression normalization^{59 (link)} as cldn7b knockout did not affect baseline expression.

mRNA was extracted from cells using the RNEasy kit (Qiagen, Hilden, Germany) and cDNA was synthesized using the Superscript IV VILO™ master mix kit (ThermoFisher Scientific, Waltham, MA, USA) according to manufacturer’s recommendations. Synthesized cDNA was them mixed with the PowerUp™ SYBR™ Green master mix kit (ThermoFisher Scientific, Waltham, MA, USA) and abundance of target transcripts detected on a CFX96™ Real-Time System (Bio-Rad, Hercules, CA, USA). Data was analyzed with the accompanying Bio-Rad CFX Manager™ software. Viral genomes were extracted using the Quick-DNA™ Miniprep Plus Kit and were detected by the same methods mentioned previously. Amplified products were analyzed by gel electrophoresis to ensure specificity. PCR primers used in this study are shown in Table 1.