Let-7 miRNA levels were profiled using a published real-time PCR-based platform57 . To summarize, 500 ng of total RNA was reverse transcribed using SuperScript IV First-Strand Synthesis System (ThermoFisher Scientific, 18091050) with primers specific to let-7a, let-7b, let-7g and U6-snRNA transcripts. For LIN28B, GAPDH and β-actin qPCR, we processed 500 ng of total RNA with iScript Reverse Transcription Supermix (Bio-Rad, 1708840). Afterwards, we performed quantitative PCR on 1 μl of cDNA using PerfeCTa SYBR Green SuperMix (Quantabio, 95053–100). qPCR primers are listed in Supplemental Table 6 . Results from qPCR were normalized to house-keeping gene to obtain ΔCT and ΔCT of samples were normalized to WT values to obtain ΔΔCT values. Relative fold-change is calculated as 2−ΔΔCT.
Perfecta sybr green supermix
Manufactured by Quantabio
Sourced in United States, United Kingdom, Germany
PerfeCTa SYBR Green SuperMix is a ready-to-use reaction mix for quantitative PCR (qPCR) that contains SYBR Green I dye, a hot-start Taq DNA polymerase, and optimized buffer components.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (24 mentions)
Qscript cdna supermix, by Quantabio (14 mentions)
Qscript cdna synthesis kit, by Quantabio (13 mentions)
Trizol, by Thermo Fisher Scientific (12 mentions)
Dnase 1, by Thermo Fisher Scientific (10 mentions)
Qscript microrna cdna synthesis kit, by Quantabio (8 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (8 mentions)
Nanodrop, by Thermo Fisher Scientific (6 mentions)
Iscript reverse transcription supermix, by Bio-Rad (5 mentions)
Protoscript 2 first strand cdna synthesis kit, by New England Biolabs (5 mentions)
Quantstudio 3, by Thermo Fisher Scientific (5 mentions)
Iscript cdna synthesis kit, by Bio-Rad (4 mentions)
Rneasy mini kit, by Qiagen (4 mentions)
Genejet rna purification kit, by Thermo Fisher Scientific (4 mentions)
Cfx96 system, by Bio-Rad (4 mentions)
Superscript 3, by Thermo Fisher Scientific (4 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (4 mentions)
Quantstudio 5 real time pcr system, by Thermo Fisher Scientific (4 mentions)
Fast sybr green master mix, by Thermo Fisher Scientific (3 mentions)
129 protocols using perfecta sybr green supermix
1
Profiling let-7 miRNA Levels by qPCR
2
Quantifying IRC7 Gene Copy Numbers
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The relative copy numbers of the IRC7 gene in selected strains were estimated with quantitative PCR of genomic DNA. Primers PF6 and PR7 from Roncoroni et al. (2011 (link)) were used for IRC7. Copy numbers were normalized to that of ALG9 and UBC6 (primers listed in Krogerus et al. (2019 (link))). The efficiencies (E) of the qPCR assays (ranging from 1.9 to 1.94) for each primer pair were calculated using the formula 10(− 1/m), where m is the slope of the line of the threshold cycle (CT)-versus-log dilution plot of the DNA template (8 pg to 8 ng input DNA) (Pfaffl 2001 (link)). The qPCR reactions were prepared with PerfeCTa SYBR® Green SuperMix (QuantaBio, Beverly, MA, USA) and 0.3 μM of the primers. The qPCR reactions were performed on a LightCycler® 480 II instrument (Roche Diagnostics, Basel, Switzerland) in four technical replicates on 1 ng template DNA. The following programme was used: pre-incubation (95 °C for 3 min), amplification cycle repeated 45 times (95 °C for 15 s, 60 °C for 30 s, 72 °C for 20 s with a single fluorescence measurement), melting curve programme (65–97 °C with continuous fluorescence measurement), and finally a cooling step to 40 °C. The copy numbers of IRC7 relative to ALG9 and UBC6 were calculated using the Pfaffl method (Pfaffl 2001 (link)).
3
Quantitative analysis of viral and host gene expression
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Total RNA was extracted from mouse lung homogenates, and viral RNA was extracted from cleared mouse lung homogenates and cell culture supernatants using the following commercially available kits according to the manufacturer’s instructions: for viral RNA extraction, QIAamp Viral RNA Mini Kit (QIAGEN) and E.Z.N.A Viral RNA kit (Omega Bio-tek) were used. Quantification of viral RNA was performed by quantitative reverse transcription PCR (RT-qPCR) as described below. For total RNA extraction, the samples were passed through a QIAshredder (QIAGEN) to reduce viscosity and subsequently RNA was isolated using the ROTI Prep RNA Mini kit (Carl Roth). After isolation, RNA concentration was measured using a NanoVue Plus Spectrophotometer (GE Healthcare). For cDNA synthesis, 0.5 µg RNA from each sample was reverse-transcribed using the qScript cDNA Synthesis Kit (Quantabio). For subsequent qPCR reactions, PerfeCTa SYBR Green SuperMix (Quantabio) was used in a total reaction volume of 15 µl. TaqMan primer/probes for mouse inflammatory genes were purchased from Microsynth and are listed below. RT-PCR was performed on a StepOnePlus Real-Time PCR System (Applied Biosystems) with the following conditions: 95°C for 3 min, followed by 45 amplification cycles (15 s at 95°C) and elongation (1 min at 60°C).
4
RNA Isolation and qPCR Analysis
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Tri-reagent (Sigma) was added to samples to lyse cells and RNA isolation was performed following the manufacturer’s instructions. cDNA was made using a cDNA reverse transcription kit (Applied Biosciences) and qPCR was performed using PerfeCTa® SYBR® Green SuperMix (QuantaBio), see Supplementary Table 2 for a list of primers used. All assays were performed in accordance with manufacturer’s instructions.
5
Quantitative RT-PCR Protocol for Gene Expression
6
SARS-CoV-2 RNA Isolation and Quantification
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Total RNA isolation from SARS-CoV-2 infected and control cells was
performed as described above. According to the manufacturer’s
instructions, RNA was reverse transcribed to cDNA using the qScript
microRNA cDNA synthesis kit (Quantabio, Lutterworth, UK), and selected
miRs were assessed by qPCR using the PerfeCTa SYBR Green SuperMix
(Quantabio, Anatolia GeneWorks Istanbul, Turkey). RNU6 was used as
a normalization reference RNA using the comparative cycle threshold
method.43 (link) Primers for each miR assessed
included hsa-miR-8066, -5197, -3611, -3934-3p, -1307-3p, -3691-3p,
and -1468-5p (Table 3 ; Sentromer DNA Technologies, Istanbul, Turkey). The following thermocycling
conditions were used: denaturation at 95 °C for 2 min, then 40
cycles of 95 °C for 2 s, 60 °C for 15 s, and extension at
72 °C for 15 s.
performed as described above. According to the manufacturer’s
instructions, RNA was reverse transcribed to cDNA using the qScript
microRNA cDNA synthesis kit (Quantabio, Lutterworth, UK), and selected
miRs were assessed by qPCR using the PerfeCTa SYBR Green SuperMix
(Quantabio, Anatolia GeneWorks Istanbul, Turkey). RNU6 was used as
a normalization reference RNA using the comparative cycle threshold
method.43 (link) Primers for each miR assessed
included hsa-miR-8066, -5197, -3611, -3934-3p, -1307-3p, -3691-3p,
and -1468-5p (
conditions were used: denaturation at 95 °C for 2 min, then 40
cycles of 95 °C for 2 s, 60 °C for 15 s, and extension at
72 °C for 15 s.
7
MicroRNA Expression Quantification by qRT-PCR
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Fifty nanograms of total RNA were reverse-transcribed using the qScript microRNA cDNA Synthesis Kit (QuantaBio) according to manufacturer instructions. Samples were analyzed through qRT-PCR using the PerfeCTa SYBR® Green SuperMix (QuantaBio) in a CFX-96 thermal cycler (Bio-Rad Laboratories) according to manufacturer instructions. All reactions were run in duplicate in a 25µL reaction. MiRNA primers were purchased from Metabion International AG (Metabion) or Sigma-Aldrich (Merck KGaA). MiR-486 was used as an endogenous normalizer. Expression levels were calculated using the 2−ΔΔCt formula.
8
RNA extraction and qRT-PCR analysis
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In order to prepare total RNA using the PeqGOLD total RNA kit (#732–2871, Peqlab/VWR Life Science, Bruchsal, Germany), 1 × 105 cells were seeded per well of a 6-well plate and treated as described in the figure legends. Subsequently, cDNA was synthesized with the qScript Flex cDNA Kit (#95049, Quantabio, Beverly, MA, USA) and used as template for qRT-PCR using the PerfeCTa SYBR Green SuperMix (#95049, Quantabio) and the CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). Relative gene expression levels were calculated by normalizing transcript levels of the genes under investigation to those of GAPDH, using the 2−ΔCt method.
9
Primer Design for Gene Expression Analysis
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The forward primer (FP) and reverse primer (RP) from coding sequences of f7, f8, αIIb, ago1, ago2, ago3a, ago3b, ago4, and rnaseh1 genes were purchased from Invitrogen, Carlsbad, CA (Table 2 ). 1 µg of total RNA was converted to cDNA at 42 °C for 30 min using qScript cDNA SuperMix (Quantabio, Beverly, MA) and qRT-PCR was performed using PerfeCTa SYBR Green SuperMix (Quantabio, Beverly, MA). The result was analyzed to determine ΔCT values from which relative fold change was measured, and the graph was plotted.
Forward and reverse primers used in qRT-PCRs
| Gene name | qRT-PCR primers (5′ to 3′) |
|---|---|
| αIIb | |
| f8 | |
| f7 | |
| ago1 | |
| ago2 | |
| ago4 | |
| ago3a | |
| ago3b | |
| rnaseh1 |
10
Quantifying miRNA Expression in iPSC-EVs
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Total microRNA (miRNA) was isolated from different iPSC-EV samples using the miRNeasy Micro Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. Reverse transcription was carried out using commercial qScript miR cDNA synthesis kit (Quantabio, Beverly, MA, USA). The PerfeCTa® Universal PCR Primer (QuantaBio) has been designed and validated to work specifically with PerfeCTa SYBR Green SuperMix using miRNA cDNA produced. The levels of miR-133, miR-155, miR-221, and miR-34a were determined. U6, SNORD48, and SNORD44 were examined as housekeeping genes for the normalization of miR expression levels (primer sequences are shown in Table 1 ). Real-time RT-PCR reactions were performed on an Applied Biosystems Quantstudio 7 flex (Applied Biosystems, Foster City, CA, USA), using SYBR1 Green PCR Master Mix (Applied Biosystems). The amplification reactions were performed as follows: 10 min at 95 °C, and 40 cycles of 95 °C for 15 s and 60 °C for 30 s, and 70 °C for 30 s. Fold variation in gene expression was quantified by means of the comparative Ct method: 2^(−(∆C_(t treatment)−∆C_(t control)), which is based on the comparison of expression of the target gene (normalized to the endogenous control) between the compared samples.
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