Gotaq qpcr master mix

miRNA quantitative PCR and data analysis were performed as described previously [34 (link),35 (link)]. Primer sequences are reported in Supplementary Table S1. Briefly, 500 ng of total RNA were reverse transcribed at 50 °C using Protoscript II Reverse transcriptase (#0368 New England Biolabs) according to the manufacturer’s protocol. As previously described, in order to avoid amplification of pre-miRNAs, RNA was not denatured before miRNA reverse transcription [34 (link),35 (link)]. cDNA was amplified by qPCR using specific primers with GoTaq^® qPCR Master Mix (#6002 Promega, Milan, Italy) in a CFX Connect instrument (BioRad).
mRNA reverse transcription was carried out at 42 °C using Protoscript II Reverse transcriptase ( New England Biolabs GmbH #0368, Frankfurt, Germany) and random hexamers (Takara Bio Europe #3801, Saint-Germain-en-Laye, France) according to the manufacturer’s protocol. qPCR amplification of mRNAs was performed using specific primers with GoTaq^® qPCR Master Mix (#6002 Promega) in a CFX Connect instrument (BioRad).

Strains ∆22915 and S2308 were cultured in TSB and collected at the exponential phase. Total RNA was extracted with TRIzol RNA isolation reagent (Ambion, Carlsbad, CA, USA). Genomic contamination was removed with Turbo DNA-free kits (Ambion). RNA was subjected to reverse transcription with PrimeScript RT reagent kits (TaKaRa) at 37 °C for 10 min, then 85 °C for 5 s for cDNA templates. GoTaq qPCR master mix (Promega, Fitchburg, WI, USA) was used for qRT-PCR, according to the manufacturer’s instruction: 1 μL cDNA, 0.5 μL forward or backward primer (10 μM), 8 μL nuclease-free water and 10 μL 2× GoTaq qPCR master mix were added. Reactions were on a Mastercycler ep Realplex system (Eppendorf, Germany) at 95 °C for 2 min, 40 cycles at 95 °C for 15 s, 60 °C for 1 min and a melting curve. Genes were tested in triplicate and the GAPDH gene was the internal control. Primers were designed according to the wild-type strain S2308 genome (GenBank Code: NC_007618.1 and NC_007624.1) with National Center for Biotechnology Information (NCBI) Primer-BLAST [24 (link)]. Relative transcription levels were calculated with the 2^−∆∆Ct method.

sqRT-PCR was performed to detect fused GFP transcripts in treated HEK293 cells. For sqRT-PCR analysis, a 5′ GFP-specific forward primer (5′-GCTGACCCTGAAGTTCATCTG-3′), a 3′ GFP-specific reverse primer (5′-CGCCGATGGGGGTATTCTGCTGG-3′), cDNA of treated cells and GoTaq^® qPCR Master Mix (Promega, Madison, WI, USA) was used. The PCR was performed using a Bio-Rad CFX™ system (BioRad, Hercules, CA, USA) with the following conditions: 95 °C for 2 min, and 50 cycles of 20 s at 95 °C, 20 s at 62 °C, and 25 s at 72 °C. Experiments were carried out in duplicates and repeated two times. Correct PCR products were verified by direct sequence analysis.
To analyze differences in cis-splicing events after transfection of HEK293 cells exon–exon junction-specific forward (Exon5/6: 5′-AGCTCAGCATGAAAGCATCCCT-3′, Exon6/7: 5′-AGGACGCCCACCTCTCCTCC-3′) and reverse (Exon6/7 5′-GGAGGAGAGGTGGGCGTCCT-3′, GFP: 5′-GGTCAGCTCGATGCGATTCACC-3′) primers, cDNA of treated cells and GoTaq^® qPCR Master Mix (Promega, Madison, WI, USA) was used. The PCR was performed using a Bio-Rad CFX™ system with the following conditions: 95 °C for 2 min, and 50 cycles of 20 s at 95 °C, 20 s at 64 °C, and 25 s at 72 °C. Experiments were carried out in duplicates and repeated two times. Correct PCR products were verified by direct sequence analysis.

Total RNA was extracted and purified using RNAiso Plus (Takara, Japan) and was further used to synthesize the first-strand cDNA using the GoScriptTM Reverse Transcription System (Promega, United States). qRT-PCR was performed on a Roche LightCycler 96, using GoTaq^® qPCR master mix (Promega, United States) with the following reaction mixture: 2 µl cDNA template, 10 µl 2 × GoTaq®qPCR master mix, 7 µl nuclease-free water, and 0.5 µl forward and reverse primers. All qRT-PCR primers were obtained from PrimerBank (https://pga.mgh.harvard.edu/primerbank/) and are listed in Supplementary Table S1. Glyceraldehyde-3-phosphate dehy drogenase (GAPDH) served as an internal control. The 2-ΔΔCt method was used to determine the relative mRNA expression level (Zhang et al., 2017 (link); Yang et al., 2020 (link); Hu et al., 2021 (link)).

Total RNA was reverse transcribed with the GoScript Reverse Transcription System (Promega, Madison, Wisconsin). Quantitative Real-Time PCR (qPCR) was performed to determine relative expression levels for goldentouch in scale tissue. After an initial denaturation step (95 °C for 2 min), qPCR reactions were run for 40 cycles (95 °C for 15 s, 60 °C for 1 min) (CFX96 Real-Time System; Bio-Rad Laboratories, Hercules, California) using specifically designed primers (goldentouch_fwd: 5′-AGC TGC TCA AAC GAC ACT GA; goldentouch_rev: 5′-ACG CCA ACC ACA ATC ACA ATG). The total volume for each reaction was 20 µl consisting of 2 µl cDNA (5 ng/µl), 0.5 µl forward primer (10 µM), 0.5 µl reverse primer (10 µM), 10 µl GoTaq qPCR Master Mix, 2× (GoTaq qPCR Master Mix; Promega, Madison, Wisconsin) and 7 µl Nuclease-free H₂O. All qPCR reactions were followed by a melt curve analysis to test for amplification specificity. Expression levels were quantified using mean threshold cycle (Ct) values based on three and two technical replicates for target genes and housekeeping genes, respectively. Relative gene expression of the target gene was calculated using the geometric mean of two housekeeping genes (ldh2_fwd: 5′-TTG GAG GTT TTG AGG AAA AGG; ldh2_rev: 5′-: CAG GAA CAA GGT GAC TGT GGT; imp2_fwd: 5′-GCC TGG AGC ATG TTG ACC; imp2_rev: 5′-CGA AGT GAC GGA TCT TAC GG).

Cardiac loops isolated from experimental and control (CFDA) embryos (Supplementary Figure S2) were subjected to qRT-PCR analysis following MIQE guidelines [72 (link),73 (link),74 (link)]. RNA was extracted and purified using the ReliaPrep RNA Cell Miniprep System Kit (Promega) according to the manufacturer’s instructions. For mRNA expression measurements, 1 μg of total RNA was used for retro-transcription with a Maxima First Strand cDNA Synthesis Kit for qRT-PCR (Thermo Scientific). Real-time PCR experiments were performed with 2 μL cDNA, Go Taq qPCR Master Mix (Promega) and corresponding primer sets (Supplementary Table S1). For microRNA expression analyses, 20 ng of total RNA was used for retro-transcription with Universal cDNA Synthesis Kit II (Exiqon) and the resulting cDNA was diluted 1/80. Real-time PCR experiments were performed with 1 μL of diluted cDNA, Go Taq qPCR Master Mix (Promega) as well. All qPCRs were performed using a CFX384TM thermocycler (Bio-Rad) following the manufacture’s recommendations. The relative expression of each gene was calculated using Gusb and Gadph as internal controls for mRNA expression analyses and 5S and 6U for microRNA expression analyses, respectively [75 (link)]. Each PCR reaction was carried out in triplicate and repeated in at least three distinct biological samples to obtain representative means.

All primers used in this study were designed using the Primer3web (RRID: SCR_003139) and NCBI Primer-BLAST (RRID: SCR_003095) online tools and are listed in Table S1. The UBIQUITIN10 (UBI10; AT4G05320) and ELONGATION FACTOR1α (EF1α; AT5G60390) transcripts were used as the reference genes to normalize RT-qPCR-generated gene expression data. The sRNAs snoR101 and U6 were used as reference sequences to normalize the quantified abundance of the miR397 sRNA after RT-qPCR. All RT-qPCR reactions were carried out on a Rotor-Gene Q machine (Qiagen, Melbourne, Australia) using GoTaq qPCR Master Mix (Promega, Sydney, Australia), and each reaction contained 1X GoTaq qPCR Master Mix, 1.0 μM of each primer, and 50 ng of cDNA template in a 10 μL total reaction volume. The cycling program for product amplification was 1 cycle of 95°C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 40 s. The melt curve was generated for each primer pair across the temperature range of 72 to 95 °C, with a temperature increment of 1.0 °C each 5 s period. Each primer pair was assessed using 4 biological replicates, and 3 technical replicates were performed per biological replicate. The resulting RT-qPCR data was analyzed by application of the delta delta Ct (^ΔΔCt) method [62 (link)].