Total RNA was extracted from human fibroblasts by using Tri reagent (Sigma, Saint Quentin Fallavier, France). The RNA pellet was resuspended in 25 μl of RNase-free distilled water and stored at −80 °C. The RNA was used for reverse transcription using Moloney murine leukemia virus (M-MLV) reverse transcriptase (Promega, Charbonnieres, France) according to the manufacturer’s instructions. The reaction was carried out using 1 μg total RNA as template for the normalization of viral RNA to the amount of total RNA. The MaximaTM Probe/ROX qPCR Master Mix (2x) (Thermo Scientific) was used in qPCR experiment. Each reaction of 25 μL contained 400 nM of each primer, 200 nM of specific probe and 1x Maxima Probe/ROX qPCR Master Mix. Primers and probes sequences are listed in Supplementary Table S1 . The amplification conditions were 95 °C for 10 min followed by 45 amplification cycles of 95 °C for 15 s, 60 °C for 20 s and 72 °C for 30 s. The reactions were performed in an Applied Biosystem 7300 system. Real time data were analyzed using the SDS software (Thermo Fischer Scientific). Viral RNA was quantified by comparing the sample’s threshold cycle (Ct) values with each virus RNA standard curve which was obtained as previously described47 (link), 54 (link).
Sds software
Manufactured by Thermo Fisher Scientific
Sourced in United States, Canada, United Kingdom
SDS software is a digital platform designed to manage and access safety data sheets (SDS) for various chemical products. It provides a centralized repository for storing, organizing, and retrieving SDS information to ensure compliance with regulatory requirements.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (21 mentions)
Rneasy mini kit, by Qiagen (19 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (15 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (12 mentions)
Rneasy plus mini kit, by Qiagen (11 mentions)
Iscript cdna synthesis kit, by Bio-Rad (10 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (7 mentions)
Abi 7500 real time pcr system, by Thermo Fisher Scientific (7 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (7 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (7 mentions)
Abi 7900ht sequence detection system, by Thermo Fisher Scientific (7 mentions)
Nanodrop, by Thermo Fisher Scientific (7 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (6 mentions)
Trizol, by Thermo Fisher Scientific (6 mentions)
M mlv reverse transcriptase, by Promega (5 mentions)
7300 real time pcr system, by Thermo Fisher Scientific (5 mentions)
Taqman snp genotyping assay, by Thermo Fisher Scientific (5 mentions)
Sybr premix ex taq, by Takara Bio (5 mentions)
7900ht real time pcr system, by Thermo Fisher Scientific (5 mentions)
Tri reagent, by Merck Group (4 mentions)
192 protocols using sds software
1
Quantification of Viral RNA in Human Fibroblasts
2
Quantitative Analysis of Cas9 and AP Transcripts
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RNA was extracted from OCT-embedded tissues using the RNeasy Fibrous Tissue kit (Qiagen). The cDNA was generated using the Super-script IV Kit (ThermoFisher Scientific) and quantified by the Qubit ssDNA assay kit (ThermoFisher Scientific) using the Qubit 3.0 Fluorometer (ThemoFisher Scientific). The Cas9 and AP transcripts were quantified by qPCR using the TaqMan Universal PCR master mix (ThermoFisher Scientific) and TaqMan custom-designed primers and probes (Supplemental Table 8 ). The qPCR was performed in the ABI 7900HT qPCR machine (ThermoFisher Scientific) using the SDS software (Version 2.4, ThermoFisher Scientific). The transcript copy number was determined by the cycle value from a quantitative reverse transcription PCR (RT-qPCR) reaction that was first converted to the raw copy number using a standard curve of the known amount of the cis-plasmid, and then divided by the total amount of cDNA (ng) used in the reaction.
3
Quantification of Viral RNA by RT-qPCR
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Total RNA was extracted using Tri reagent (Sigma, Saint Quentin Fallavier, France) according to the manufacturer’s protocol. The RNA pellet was resuspended in 25 μL of RNase-free distilled water and stored at −80 °C. The RNA was used for reverse transcription using Moloney murine leukemia virus (M-MLV) reverse transcriptase (Promega, Charbonnieres, France) according to the manufacturer’s instructions. The reaction was carried out using 1 μg total RNA as template for the normalization of viral RNA to the amount of total RNA. The MaximaTM Probe/ROX qPCR Master Mix (2×) (Thermo Scientific) was used in qPCR experiment. Each reaction of 25 μL contained 400 nM of each primer, 200 nM of specific probe and 1× MaximaTM Probe/ROX qPCR Master Mix. Primers and probe sequences are listed in Table S4 . The amplification conditions were 95 °C for 10 min followed by 45 amplification cycles of 95 °C for 15 s, 60 °C for 20 s and 72 °C for 30 s. The reactions were performed in an Applied Biosystem 7300 system. Real time data were analyzed using the SDS software (Thermo Fischer Scientific). Viral RNA was quantified by comparing the sample’s threshold cycle (Ct) values with each virus RNA standard curve which was obtained as previously described [20 (link),21 (link)].
4
Quantifying EV-miRNAs hsa-miR-30e-3p and hsa-miR-103a-3p
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Expression quantification of hsa-miR-30e-3p and hsa-miR-103a-3p EV-miRNAs was determined by Custom TaqMan™ microRNA assay (Thermo Fisher Scientific, Waltham MA, USA) following standard procedures. Briefly, each miRNA was analyzed in triplicate, and RNU48 was used for data normalization. Specific reverse transcription of miRNAs was performed following standard procedures. R.T. was performed using a C1000 Thermal Cycler (Bio-Rad, Hercules, CA, USA), and the cDNAs were pre-amplified. After the pre-amplification product was diluted at 1:8, quantitative RT-PCR was run in a Quant Studio 12K Flex Fast Real-Time PCR System (Thermo Fisher Scientific), according to the manufacturer’s protocol. miRNA expression was calculated by the comparative cycle threshold (ΔCT) method and analyzed with SDS software (Thermo Fisher Scientific).
5
Quantitative Analysis of Pigmentation Genes
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Total RNA was reverse transcribed into cDNA using the Quantitect Reverse Transcription kit (Qiagen). Quantitative PCR (qPCR) was performed in SYBR Green master mix (Qiagen) with an Applied Biosystems 7500 PCR and analyzed with the SDS software (Keenen et al., 2010 (link)) (Thermo Fisher). The mRNA levels of human pigmentation genes MITF, SOX10, TYR, TYRP1, DCT, and IRF4 were normalized to ACTβ. Corresponding murine mRNA levels of Mitf, Sox10, Tyr, Tyrp1, and Dct were normalized to Rpl7. Oligonucleotide primer sequences are listed in Table S1 .
6
Quantitative RT-PCR for Viral RNA Quantification
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Total RNA was extracted from human fibroblasts by using Tri reagent (Sigma-Aldrich). The RNA pellet was resuspended in 25 μl of RNase-free distilled water and stored at -80 °C. The RNA was then used for reverse transcription using MMLV reverse transcription Kit (Promega) according to the manufacturer's instructions. The reaction was carried out using 1 μg total RNA as template for the normalization of viral RNA to the amount of total RNA. The MaximaTM Probe/ROX qPCR Master Mix (2x) (Thermo Scientific) was used in qPCR experiments. Each reaction of 25 μL contained 400 nM of each primer, 200 nM of specific probe and 1x Maxima Probe/ROX qPCR Master Mix.
Primers and probe sequences are listed in Table 1(Tab. 1) . The amplification conditions were 95 °C for 10 min followed by 45 amplification cycles of 95 °C for 15 s, 60 °C for 20 s and 72 °C for 30 s. The reactions were performed in an Applied Biosystem 7300 system. Real-time data were analyzed using the SDS software (Thermo Fisher Scientific). Viral RNA was quantified by comparing the sample's threshold cycle (Ct) values with each virus RNA standard curve which was obtained as previously described (Wichit et al., 2017[28 (link)]).
Primers and probe sequences are listed in Table 1
7
Genotyping of SNPs rs11081062 and rs26728
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Venous blood samples from patients and controls were collected, and DNA was extracted by standard procedures. TaqMan genotyping was performed for SNPs rs11081062 and rs26728 using the following reactions: 5 U/L super mix, 0.9 ng forward primer, 0.9 ng downstream primer, 0.5 ng FAM (6-carboxyfluorescein), 0.5 ng VIC (4,7,2′-trichloro-7′-phenyl-6-carboxyfluorescein), and 1 U/L genomic DNA. PCR was conducted in an Applied Biosystems® 7500 real-time PCR system with the following conditions: 95°C for 3 minutes, followed by 40 cycles of 95°C for 15 seconds, and 60°C for 1 minute. Positive and negative controls were included with all reactions. Genotype data were interpreted using SDS software (Thermo Fisher Scientific, Waltham, MA, USA).
8
Quantification of Chikungunya Virus RNA
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Total RNA was extracted from SLC, MB and MT using Tri Reagent (Sigma-Aldrich). RNA was reverse transcripted using Moloney murine leukemia virus (M-MLV) reverse transcriptase (Promega, Charbonnieres, France) according to the manufacturer's instructions. The reaction was carried out using 1 mg total RNA as template for the normalization of viral RNA to the amount of total RNA. The Maxima TM Probe/ROX qPCR Master Mix (2x) (Thermo Fisher Scientific, Illkirch, France) was used in qPCR experiment. Each reaction of 25 mL contained 400 nM of each primer, 200 nM of specific probe and 1x Maxima Probe/ROX qPCR Master Mix. The following CHIKV Primer Sequence (5'->30) were used:
CHIKV-P 50CCAATGTC (TC)TC (AC)GCCTGGACACCT3' [39] (link).
The amplification conditions were 95 °C for 10 min followed by 45 amplification cycles of 95°C for 15 s, 60 °C for 20 s and 72 °C for 30 s. The reactions were performed in an Applied Biosystem 7300 system. Real time data were analyzed using the SDS software (Thermo Fischer Scientific). Viral RNA was quantified by comparing the sample's threshold cycle (Ct) values with each virus RNA standard curve that was obtained as previously described [39] (link). Data are expressed as mean ± SEM of two independent experiments performed in triplicate.
CHIKV-P 50CCAATGTC (TC)TC (AC)GCCTGGACACCT3' [39] (link).
The amplification conditions were 95 °C for 10 min followed by 45 amplification cycles of 95°C for 15 s, 60 °C for 20 s and 72 °C for 30 s. The reactions were performed in an Applied Biosystem 7300 system. Real time data were analyzed using the SDS software (Thermo Fischer Scientific). Viral RNA was quantified by comparing the sample's threshold cycle (Ct) values with each virus RNA standard curve that was obtained as previously described [39] (link). Data are expressed as mean ± SEM of two independent experiments performed in triplicate.
9
Absolute Quantification of CMV and GAPDH
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For absolute quantification of cytomegalovirus and GAPDH copy numbers, one plasmid containing both target sequences served as external standard (Thermo Fisher Scientific). Tenfold dilution series ranging from 2 × 10 6 to 2 × 10 2 copies were used to generate two standard curves by plotting the Ct values to the copy numbers (Supplementary Figure S1). Calculation of absolute cytomegalovirus and GAPDH copy numbers in unknown samples was achieved via the SDS software (version 1.3.1, Thermo Fisher Scientific) by referring to the respective standard curve. As GAPDH is a single copy gene with two alleles per diploid genome, half the amount of GAPDH copy numbers equals analyzed cell number. Finally, viral load was expressed as cytomegalovirus copy numbers/10 5 cells. All samples and standards were analyzed at least in duplicates. Amplification of GAPDH was also used to control absence of PCR inhibitors in extracted DNA samples. The mean GAPDH-Ct value of 27.6 ± 0.9 that is derived from 115 samples served as a guide level. Samples with GAPDH-Ct-values ≥ 30.6 were excluded or re-analyzed. Assessment of cytomegalovirus copy numbers using the Qiagen Artus Kit was carried out according to the manufacturer's instructions (Qiagen).
10
DNA Extraction and Genotyping Protocol
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Genomic DNA was extracted using the TIANamp Blood DNA Kit (TianGen Biotech Co. Ltd., Beijing, China) according to the manufacturer’s instructions. TaqMan real-time polymerase chain reaction (PCR) was applied to acquire the genotypes by using the ABI QuantStudio DX system [15 (link),16 (link)]. Briefly, the final volume was 10 μl and consisted of 5 μl TaqMan universal PCR Master Mix, 0.5 μl Primer/probe mix and 10 ng genomic DNA. The PCR program had an initial denaturation step at 95°C for 10 min and 40 cycles at 94°C for 15 s and 60°C for 45 s. Allele frequencies were analyzed by using ABI SDS software. For quality control, genotyping was repeated on a random 10% of the samples, and 100% concordance rate was observed for the results.
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