Journal: bioRxiv

Article Title: Synthetic circRNAs employ IRES activity for translation in cells and in cell-free translation systems

doi: 10.64898/2026.03.28.715045

Figure Lengend Snippet: (A) Experimental outline of the in vitro generated circRNA-based reporter assay based on the mRuby3_circ_EGFP_ScaI_v3-4 plasmid (pKL480) for evaluation of IRES activity of different IRES inserts, including inverse sequences as controls for circRNA translation dependent on insert length and GC-content. Following DNA template synthesis (PCR amplification from plasmid backbone using poly(A)- and poly(T)-including primers), circRNA was generated by T7 in vitro transcription. Linear RNA species were removed by RNase R-treatment and column purification. Resulting circRNA was subsequently used for in-cell transfection or for in vitro translation systems. EGFP and 3xHA-Nluc reporter systems were used throughout the following analyses. The plasmid-encoded mRuby was not used. (B) Illustration of the circularization reaction mediated by group I dt introns leading to self-spliced circRNA. The observed sequence scar formed by the remaining parts of the td introns are highlighted in light and dark red. Remaining split introns were generated as side products and need to be removed. (C) Overview of the tested IRES sequences indicating length and viral or cellular origin of the IRESes. (D) Quality control of the generated EGFP reporter including circRNAs using 1% FA-agarose gel. Linear side products (upper band) disappear by RNase R digestion. Only the circular RNase R-resistant band remains (lower band). 500 ng total RNA was loaded per lane. The RiboRuler High Range RNA Ladder (Thermo, #SM1821) serves as a reference. (E) Quality control of the generated 3xHA-Nluc encoding circRNAs using 1% FA-agarose gel. Linear side products (upper band) are mainly degraded by RNase R digestion. Only the circular RNase R-resistant band remains (lower band). Only Dlx1 shows less efficient circularization leading to a higher concentration of side products. Therefore, the RNase R incubation time was increased to 90 min. 500 ng total RNA was loaded per lane. RNA species: linear (L), circular (C), introns (I). (F) Quality control, using the High Sensitivity RNA ScreenTape, of the generated circRNA before purification (upper panel) and after RNase R digestion (lower panel). Remaining contaminants can be observed as light grey bands.

Article Snippet: The PCR products were purified using the Monarch PCR & DNA Cleanup kit (NEB, T1030L) and 1 μg DNA was subsequently used for in vitro transcription using the High Scribe T7 High Yield RNA synthesis Kit (NEB, E2040S) according to manufacturer’s protocol (20 μL total reaction volume) supplemented with 40 U RiboLock RNase inhibitor (Thermo, EO0381) and incubated for 2 hrs at 37°C and 800 rpm.

Techniques: In Vitro, Generated, Reporter Assay, Plasmid Preparation, Activity Assay, Amplification, Purification, Transfection, Sequencing, Control, Agarose Gel Electrophoresis, Concentration Assay, Incubation

Journal: Biotechniques

Article Title: Improved SARS-CoV-2 PCR detection and genotyping with double-bubble primers

doi: 10.2144/btn-2021-0063

Figure Lengend Snippet: (A–C) Dose–response curves using double-bubble primer mix #8 and VIC-TqM probe #12 and decimal dilutions of SARS-CoV-2 synthetic RNA N. (A) Real-time amplification plot. C t values: 8 × 10 5 copies, 22.82; 8 × 10 4 copies, 26.27; 8 × 10 3 copies, 30.55; 8 × 10 2 copies, 32.51. (B) 5% agarose gel of the PCR amplification products. Lane 1: ultra-low-range ladder; lanes 2–5: 139-bp amplicons of 8 × 10 5 to 8 × 10 2 RNA copies per tube. Also included in the PCR reaction mix were 2× TqM buffer without UDP and iScript reverse-transcriptase. (C) Efficiency plot of the PCR reaction depicted in panel A. Slope = -3.3359 represents PCR reaction efficiency of 99.42%; R 2 = 0.9816. (D) One-tube RT-qPCR using double-bubble primer mix #10 from region 2 using SARS-CoV-2 synthetic RNA (2 × 10 4 copies per tube) and FAM-TqM probe #13. RT-qPCR was performed with standard conditions.

Article Snippet: For cDNA synthesis, iScript reverse-transcriptase kit was employed: 25°C, 5 min, 42°C, 15 min 95°C, 1 min, 4°C hold (BioRad, 1708891).

Techniques: Amplification, Agarose Gel Electrophoresis, Reverse Transcription, Quantitative RT-PCR

Journal: Biotechniques

Article Title: Improved SARS-CoV-2 PCR detection and genotyping with double-bubble primers

doi: 10.2144/btn-2021-0063

Figure Lengend Snippet: (A) qPCR using reverse-transcribed cDNA template and double-bubble (D-B) primer mix #10, FAM-TqM probe #13, TqM fast kit+ uracil-DNA glycosylase (UDG) assayed in duplicates. Insert depicts 5% agarose gel; lane 1: ultra-low-range (ULR) ladder; lanes 2 & 3: template cDNA; lanes 4 & 5: non-template control (NTC). C t average values: cDNA, 28.60; NTC, undetermined. (B) One-tube RT-qPCR using SARS-CoV-2 synthetic RNA N template and D-B primer mix #8, VIC-TqM probe #12, TqM fast kit without UDG iScript reverse transcriptase, assayed in duplicate. Insert depicts 5% agarose gel; lane 1: ULR ladder; lanes 2 and 3: template RNA; lanes 4 and 5: NTC. C t average values: RNA, 24.63, NTC, undetermined. (C) Duplex qPCR using SARS-CoV-2 synthetic RNA N template and TqM fast kit with UDG. C t values: primer 8, 26.13; primer 10, 32.19; duplex primers 8 + 10, 26.36 and 30.27, respectively; NTC, undetermined. (D) 5% agarose gel of samples shown in panel C. Lane 1: ULR ladder; lane 2: primer D-B mix #8 (amplicon 139 bp); lane 3: D-B primer mix #10 (amplicon 158 bp); lane 4: duplex of both primers #8 and #10 (amplicons 139 and 158 bp). Arrows (C & D) show duplex amplifications in the same tube with D-B primers #8 and #10. PCR was performed using fast conditions with 40 (A, C & D) and 30 (B) cycles.

Article Snippet: For cDNA synthesis, iScript reverse-transcriptase kit was employed: 25°C, 5 min, 42°C, 15 min 95°C, 1 min, 4°C hold (BioRad, 1708891).

Techniques: Reverse Transcription, Agarose Gel Electrophoresis, Control, Quantitative RT-PCR, Amplification

Journal: Biotechniques

Article Title: Improved SARS-CoV-2 PCR detection and genotyping with double-bubble primers

doi: 10.2144/btn-2021-0063

Figure Lengend Snippet: The reaction mixture contained hot-start double-bubble (D-B) primer mix #4, VIC-TqM probe #12, cost-effective non-hot-start Taq polymerase (FroggaBio), reverse transcriptase (iScript), synthetic SARS-CoV-2 RNA gene N as template, added dNTPs and ROX dye for internal calibration. The reaction was assembled at room temperature and subjected to real time RT-qPCR using the following fast conditions: 42°C, 15 min; 95°C,1 min; 30 cycles of 95°C, 1 s → 70°C, 20 s. The amplification plot and (insert) 5% agarose gel depict the amplification of SARS-CoV-2 RNA extracted from the nasopharyngeal swabs of patient S1 (lane 2) and negative patient N1 (lane 3), no-template controls (lanes 4 and 5) and SARS-CoV-2 gene N synthetic RNA as positive control (lane 6). Note the amplification in the real-time plot and the 115-bp band in the 5% agarose gel with SARS-CoV-2 virus or synthetic RNA (lanes 2 and 6), with no amplifications in the negative control N1 (lane 3) or NTC (lanes 4 and 5). Lane 1: ultra-low-range ladder. C t values: positive patient S1: 23.95; synthetic RNA: 23.84; negative patient N1 and NTC: undetermined.

Article Snippet: For cDNA synthesis, iScript reverse-transcriptase kit was employed: 25°C, 5 min, 42°C, 15 min 95°C, 1 min, 4°C hold (BioRad, 1708891).

Techniques: Reverse Transcription, Quantitative RT-PCR, Amplification, Agarose Gel Electrophoresis, Positive Control, Virus, Negative Control