Superscript vilo cdna synthesis kit

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany, United Kingdom, Canada, Australia, Japan, Italy, China, France, Belgium, Finland, Cameroon

The SuperScript VILO cDNA Synthesis Kit is a versatile tool for reverse transcription of RNA to complementary DNA (cDNA). It uses a proprietary enzyme system to efficiently convert RNA into cDNA, which can then be used in downstream applications such as real-time PCR, gene expression analysis, and cloning.

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CDNA synthesis kit (1264 citations) SuperScript VILO (354 citations) SuperScript VILO kit (163 citations) SuperScript cDNA synthesis kit (97 citations) SuperScript VILO cDNA Kit (40 citations) VILO kit (25 citations) CDNA kits (3 citations)

2 145 protocols using superscript vilo cdna synthesis kit

Quantitative PCR Analysis of Skin Cells

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RNA was purified from FACS sorted cells by directly sorting into TrizolLS (Invitrogen) and purified using Direct-zol RNA MiniPrep kit (Zymo Research). Equivalent amounts of RNA were reverse-transcribed by SuperScript VILO cDNA Synthesis Kit (Invitrogen). cDNAs were normalized to equal amounts using primers against β-actin. cDNAs were mixed with indicated primers and Power SYBR Green PCR Master Mix (Applied Biosystems), and quantitative PCR (qPCR) was performed on a Applied Biosystems 7900HT Fast Real-Time PCR system. Primer sequences for RT-PCR were obtained from Roche Universal ProbeLibrary.
For Vγ5 qPCR, unwounded and wounded skin was incubated in 50mM EDTA for 1hour, to separate epidermis was separated from dermis. Epidermal cells were immediately frozen in liquid nitrogen. Frozen tissues were homogenized using Bessman Tissue Pulverizer (SpectrumTM) and collected in Trizol (Invitrogen). RNA was extracted using Direct-zol RNA MiniPrep kit (Zymo Research) per manufacturer’s instructions. Equivalent amounts of RNA were reverse-transcribed by SuperScript VILO cDNA Synthesis Kit (Invitrogen). cDNAs were mixed with indicated primers and Power SYBR Green PCR Master Mix (AppliedBiosystems), and quantitative PCR (qPCR) was performed on a Applied Biosystems 7900HT Fast Real-Time PCR system.

Keyes B.E., Liu S., Asare A., Naik S., Levorse J., Polak L., Lu C.P., Nikolova M., Pasoli H.A, & Fuchs E. (2016). Impaired Epidermal to Dendritic T-Cell Signaling Slows Wound Repair in Aged Skin. Cell, 167(5), 1323-1338.e14.

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Transcriptome Analysis by Ion AmpliSeq

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Total RNA was purified using an RNeasy Micro Kit (Qiagen) and treated with the DNase-one Kit (Qiagen) to remove genomic DNA. Briefly, 10 ng of total RNA was transcribed to obtain single-stranded cDNA using the SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific). A cDNA library was synthesized using the Ion AmpliSeq Transcriptome Human Gene Expression Core Panel (Thermo Fisher Scientific) and Ion Ampliseq Library Kit Plus (Thermo Fisher Scientific) according to the manufacturer’s protocol. Barcode-labeled cDNA libraries were analyzed by the Ion S5 XL System (Thermo Fisher Scientific) using the Ion 540 Chip Kit (Thermo Fisher Scientific). Total RNA was purified using the RNeasy Micro Kit (Qiagen) and treated with the DNase-one Kit (Qiagen) to remove genomic DNA. We reverse transcribed 10 ng of total RNA to obtain single-stranded cDNA using the SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific). We performed cDNA library synthesis using the Ion AmpliSeq Transcriptome Human Gene Expression Core Panel (Thermo Fisher Scientific) and the Ion Ampliseq Library Kit Plus (Thermo Fisher Scientific) according to the manufacturer’s protocol. Barcode-labeled cDNA libraries were analyzed by the Ion S5 XL System (Thermo Fisher Scientific) using the Ion 540 Chip Kit (Thermo Fisher Scientific).

Mitsuzawa S., Zhao C., Ikeguchi R., Aoyama T., Kamiya D., Ando M., Takeuchi H., Akieda S., Nakayama K., Matsuda S, & Ikeya M. (2020). Pro-angiogenic scaffold-free Bio three-dimensional conduit developed from human induced pluripotent stem cell-derived mesenchymal stem cells promotes peripheral nerve regeneration. Scientific Reports, 10, 12034.

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Astrocyte RNA Extraction and qPCR Analysis

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Primary astrocytes were washed with PBS and then incubated for 10 min with RNA lysis buffer. RNA extraction was achieved using the Agilent Nanoprep kit according to the manufacturer’s instructions. Complementary DNA (cDNA) was generated using the SuperScriptTM VILOTM cDNA synthesis kit (Invitrogen). The FastStart Universal SYBR Green Master mix (ROX; Roche) was used on a QuantStudio 5 Real-Time polymerase chain reaction (PCR) system. Primers used were as follows: Hmgcs1 (5′-ATGGGGCTCGTGCATAGTAA-3′ and 5′-ACTCTCAGTGCTCCCCGTTA-3′); Fdps (5′-GCACTGACATCCAGGACAAC-3′ and 5′-AGCCACTTTTTCTGGGTCCT-3′); Mvk (F: 5′-CTCAAGGACGGGGTCTCC−3’ and R: 5’-GGCCCACTTGTTGATTGACT-3’); Fdft1 (5′-TCCCTGACGTCCTCACCTAC-3′ and 5′-CCCCTTCCGAATCTTCACTA-3′); Nfe2l2 (5′-CAGCTCAAGGGCACAGTGC-3 and 5′-GTGGCCCAAGTCTTGCTCC-3′); Gclc (5’-CCAACCATCCGACCCTCTG-3’ and 5’-TGTTCTGGCAGTGTGAATCC-3’); Nqo1 (5’-CCTTCCGAGTCATCTCTAGC-3’ and 5’-AGCAAGGTCTTCTTATTCTGGA-3’); Gapdh (F: 5′-GGGTGTGAACCACGAGAAAT-3’ and R: 5′-CCTTCCACAATGCCAAAGTT-3′); Aldh1l1 (F: 5’- CTTTGACCTTGGGTGCCT-3’ and R: 5’-ATCTGCTTTCCCATCCTTGT-3’).

Molina-Gonzalez I., Holloway R.K., Jiwaji Z., Dando O., Kent S.A., Emelianova K., Lloyd A.F., Forbes L.H., Mahmood A., Skripuletz T., Gudi V., Febery J.A., Johnson J.A., Fowler J.H., Kuhlmann T., Williams A., Chandran S., Stangel M., Howden A.J., Hardingham G.E, & Miron V.E. (2023). Astrocyte-oligodendrocyte interaction regulates central nervous system regeneration. Nature Communications, 14, 3372.

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Quantitative Analysis of Angiogenic Factors

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Total RNA was extracted with TRIzol reagent (Life Technologies). Reverse transcription was carried out using the RT-PCR kit (SuperScript^TM VILO^TM cDNA synthesis kit, Invitrogen, Carlsbad, CA, USA). Real-time PCR was performed using LightCycler technology with SYBR green I dye (Roche Diagnostics, Mannheim, Germany) or Universal Master mix II (Applied Biosystems, Foster City, CA, USA). Commercial VEGF-A (Hs00900055), VEGF-B (Hs00173634), PDGF-A (Hs00234994), PDGF-B (Hs00966522), FGF-1 (Hs01092738), FGF-2 (Hs00266645), EGF (Hs01099990), HGF (Hs00300159), and glyceraldehyde phosphate dehydrogenase (GAPDH) (Hs02758991) (Applied Biosystems) primer sets were used for PCR amplification under the conditions recommended by the manufacturer. For PCR amplification of IL-8, commercial primer sets (Roche Search LC, Heidelberg, Germany) were used under conditions recommended by the manufacturer. GAPDH served as an internal reference gene. The relative mRNA expression levels of host genes divided by the amount of GAPDH mRNA were evaluated statistically.

Watanabe T., Ninomiya H., Saitou T., Takanezawa S., Yamamoto S., Imai Y., Yoshida O., Kawakami R., Hirooka M., Abe M., Imamura T, & Hiasa Y. (2020). Therapeutic effects of the PKR inhibitor C16 suppressing tumor proliferation and angiogenesis in hepatocellular carcinoma in vitro and in vivo. Scientific Reports, 10, 5133.

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Nanoparticle Exposure and Radiation Effects

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16HBE14o- cells were treated or not with the NMs at 16 or 32 µg/cm², combined or not with 4 Gy irradiation. Cell pellets were collected immediately (T0), 24, 48 or 72 h after treatment. RNA extractions were realized with RNA InstaPure^TM System (Eurogentec, Liège, Belgique). After homogenization of the cell pellets with 1 mL of RNA Instapure, RNA was extracted by the addition of a 0.2 volume of chloroform, then precipitated with 1 volume of isopropanol, and finally washed 2 times with 70% ethanol as per manufacturer’s instructions. RNA quantity was measured by a NanoDrop (Thermo Fisher) and RNA quality was confirmed by agarose gel migration. cDNA synthesis was realized using the SuperScript^TM VILO^TM cDNA Synthesis Kit (Invitrogen, Carlsbad, CA, USA) as instructed per the manufacturer’s instructions. PCR probes for HMOX detection (Hs01110250_m1), NQO1 detection (Hs02512143_s1) and TXNRD1 detection (Hs00917067) were provided by Applied Biosystems (Foster city, CA, USA). RT-PCR was realized on a 7300 Real Time PCR System (Applied Biosystems, Foster city, CA, USA).

Wang Y., Sauvat A., Lacrouts C., Lebeau J., Grall R., Hullo M., Nesslany F, & Chevillard S. (2020). TiO2 Nanomaterials Non-Controlled Contamination Could Be Hazardous for Normal Cells Located in the Field of Radiotherapy. International Journal of Molecular Sciences, 21(3), 940.

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Brain gene expression analysis

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Thirty minutes after the dishabituation test, fish were sacrificed by putting them in a bath of ice-cold phosphate-buffered saline solution (PBS; Fisher Bioreagents, USA). Then, their brains were dissected and the expression of c-fos and egr-1 was analyzed.
Telencephalon, thalamus, optic tectum, retina, cerebellum and medulla oblongata were collected and used for total RNA extraction. Total RNA was extracted using the QIAGEN RNeasy Mini Kit (QIAGEN, Germany) according to manufacturer’s instructions. Briefly, brain tissues were homogenized in lysis buffer, centrifuged and then loaded onto RNeasy spin columns. Samples were treated with DNase (RNase-Free DNase Set; QIAGEN, Germany) in order to prevent genomic DNA contamination. After a series of washes and centrifugations, total RNA was eluted from columns and quantified using the Nanodrop^TM spectrophotometer (Nanodrop^TM One^C; ThermoFisher Scientific, USA). Reverse transcription was performed using the SuperScript^TM VILO^TM cDNA Synthesis Kit (Invitrogen, ThermoFisher Scientific, USA) according to manufacturer’s instructions.

Messina A., Potrich D., Schiona I., Sovrano V.A., Fraser S.E., Brennan C.H, & Vallortigara G. (2020). Response to change in the number of visual stimuli in zebrafish:A behavioural and molecular study. Scientific Reports, 10, 5769.

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Tissue Extraction and DNA/RNA Isolation

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DNA was extracted from blood using the Nucleon BACC3 Kit (GE Healthcare) and from saliva using the Oragene DNA Kit (DNA Genotek) according to the manufacturers’ directions. FFPE tissue blocks were sectioned to 8 μm, dewaxed and rehydrated using a standard xylene-ethanol deparaffinisation protocol. Tumour and benign glands were marked on haematoxylin and eosin stained tissue sections by a pathologist (SD, RM). Marked tumour and benign regions were macro-dissected separately for both DNA and RNA. DNA was extracted using the QIAamp DNA FFPE Tissue Kit (QIAGEN) according to the manufacturer’s instructions and eluted in 50 μL of ATE Buffer. DNA was quantified using the Nanodrop® ND-1000 UV-vis spectrophotometer (Nanodrop® Technologies). RNA was extracted using the RecoverAll Total Nucleic Acid Isolation Kit (ThermoFisher Scientific) according to the manufacturer’s instructions and eluted in 30 μL of dH₂O. RNA quality and quantity was assessed using the 2100 Bioanalzyer (Agilent). The SuperScript^TM VILO^TM cDNA Synthesis Kit (Invitrogen) was used for cDNA conversion, as per the manufacturer’s instructions.

FitzGerald L.M., Raspin K., Marthick J.R., Field M.A., Malley R.C., Thomson R.J., Blackburn N.B., Banks A., Charlesworth J.C., Donovan S, & Dickinson J.L. (2017). Impact of the G84E variant on HOXB13 gene and protein expression in formalin-fixed, paraffin-embedded prostate tumours. Scientific Reports, 7, 17778.

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cDNA Synthesis from Extracted RNA

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cDNA was synthesized from the extracted RNA using the SuperScript TM VILO TM cDNA Synthesis kit (Invitrogen, Thermo Fisher). The cDNAs obtained from all paired samples were stored at -20°C until real-time polymerase chain reaction (PCR) was performed.

Chazan F.L., Bonetti T.C., Gomes M.T., Fornazari V.A., Girão M.J, & Bonduki C.E. (2021). Extracellular matrix metalloproteinase expression in endometrial tissue after arterial embolization of myomas. Clinics, 76, e2145.

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RNA Extraction and qRT-PCR Analysis

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Total RNA from prepared samples was extracted using miRNeasy kit (Qiagen). Extracted RNA was then added to cDNA synthesis mix (SuperScriptTM VILOTM cDNA Synthesis Kit, Invitrogen) composed of 1× SuperScriptTM Enzyme Mix and 1× VILO™ Reaction Mix. cDNA was prepared with the following incubation protocol: 25 °C, 10 min; 42 °C, 60 min; 80 °C, 5 min for enzyme inactivation. As-prepared cDNA was added to qRT-PCR reaction mix composed of 1× TaqManTM Fast Advanced Master Mix (ThermoFisher Scientific) and 1× gene-specific TaqManTM Gene Expression Assay Mix (ThermoFisher Scientific). qRT-PCR was conducted on CFX Opus 96 real-time PCR instrument (Bio-Rad) with the following steps: 95 °C, 1 min and then subsequent thermal cycling schedule (55 cycles): 95 °C, 3 sec; 60 °C, 30 sec; fluorescence measurement. Setting the fluorescent threshold value to 400, quantification cycle (C_q) values were determined by the system software.

Woo H.K., Cho Y.K., Lee C.Y., Lee H., Castro C.M, & Lee H. (2022). Characterization and modulation of surface charges to enhance extracellular vesicle isolation in plasma. Theranostics, 12(5), 1988-1998.

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qRT-PCR Workflow for Gene Expression

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Cell counts were determined and normalized across all samples before lysis. RNA was extracted using the RNeasy Mini kit (Qiagen). After the cell lysis step, samples were spiked-in with the ERCC spike-in controls (2 µL/sample, diluted 1:100, Invitrogen #4456740) and treated with on-column DNase digestion according to the manufacturer’s specifications (Qiagen). RNA purity and concentration were measured by absorbance at 260nm (A₂₆₀/A₂₈₀). cDNAs were reverse-transcribed using the SuperScript^TM VILO^TM cDNA Synthesis kit (Invitrogen) with 100 ng to 1 µg of RNA template as directed by the manufacturer’s protocol. qRT-PCRs were carried out in triplicates using the TaqMan assay (Applied Biosystems) and CFX96 or CFX384 Touch Real-Time PCR Detection System according to manufacturers’ recommendations (Bio-Rad). Average cycle threshold (C_t) values were calculated for each gene, and the maximum C_t value was set at 40 cycles. Average C_t values of technical replicates were normalized to the exogenous spike-in or reference gene, ERCC-00096 or GAPDH respectively, and relative gene expression was determined using the comparative ΔΔC_t method. Average and SD were results of at least three independent experiments. Specific TaqMan gene expression assay IDs were as follows: ERCC-00096 (Ac03459927_a1), GAPDH (Hs02786624_g1), and MYC (Hs99999003_m1).

Ang H.X., Sutiman N., Deng X.L., Liu A., Cerda-Smith C.G., Hutchinson H.M., Kim H., Bartelt L.C., Chen Q., Barrera A., Lin J., Sheng Z., McDowell I.C., Reddy T.E., Nicchitta C.V, & Wood K.C. (2023). Cooperative regulation of coupled oncoprotein synthesis and stability in triple-negative breast cancer by EGFR and CDK12/13. Proceedings of the National Academy of Sciences of the United States of America, 120(38), e2221448120.

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