Superscript 3 rt pcr kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Superscript III RT-PCR kit is a reverse transcription-polymerase chain reaction (RT-PCR) system designed for sensitive and efficient detection of RNA targets. The kit includes a thermostable reverse transcriptase enzyme and a high-fidelity DNA polymerase for reliable cDNA synthesis and amplification.

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43 protocols using superscript 3 rt pcr kit

Quantitative RT-PCR Analysis of Autophagy Genes

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RNA was extracted from control and drug-treated cells using TRIzol as specified by the manufacturer's protocol. The RNA was treated with DNase (DNase I-RNase-Free; Ambion; Thermo Fisher Scientific, Inc.) to remove any contaminating DNA; 200 ng of total RNA was reverse-transcribed with oligo dT primers using the SuperScript^™ III RT-PCR kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) in a 20 µl reaction, as specified by the manufacturer's protocol. For quantitative PCR, the template cDNA was added to a 20 µl reaction with Power SYBR-Green PCR Master mix and 0.2 µM of primers for the target genes and GAPDH.
The primer sequences were as follows: LC3A forwards, GCC TTT CAA GCA GCG GCG GAG C, and reverse, TTG GTC TTG TCC AGG ACG GGC A; LC3B forwards, CAG CGT CTC CAC ACC AAT CTC A, and reverse, AAT TTC ATC CCG AAC GTC TCC T; Beclin-1 forwards, CTC CAT TAC TTA CCA CAG CCC A, and reverse, GGA TGA ATC TGC GAG AGA CAC C; GAPDH forwards, GCA AAT TCC ATG GCA CCG T, and reverse, TCG CCC CAC TTG ATT TTG G.
Amplification was performed with a Prism 7000 machine (Applied Biosystems; Thermo Fisher Scientific, Inc.), with the following conditions: Initial denaturation at 95°C for 10 min, then 40 cycles of 95°C for 15 sec followed by 60°C for 1 min. The fold changes relative to GAPDH were calculated using the 2^−∆∆Cq method (17 (link)).

Wang Y., Gao H., Wu T., Wang Z., Song F., Chen A., Zhang J., Zhang W., Zhang H, & Yu J. (2017). Pseudolaric acid B induced autophagy, but not apoptosis, in MRC5 human fibroblast cells. Oncology Letters, 15(1), 863-870.

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Transcription Factor Expression Profiling

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Whole blood was obtained by venous puncture and stored in PAXgene tubes (BD Biosciences) at -80°C, for a period below six months. Whole RNA was prepared using PAXgene Blood RNA Kit (Qiagen) according to the manufacturer’s instructions. RNA was quantified on a Nanodrop ND-1000 spectrophotometer (Nanodrop, Wilmington, DE, USA). cDNA synthesis carried out using the Superscript III RT-PCR kit (Applied Biosystems, Branchbug, NJ, USA). Taqman real time PCRs were performed via the universal PCR Master Mix (x2) and specific primers and probes (Applied Biosystems). Briefly, PCR was performed in the ABI Prism 7000 sequence detection system (Applied Biosystems) at 50°C for 2 min, 95°C for 10 min, 45 cycles of 95°C for 15 s, and 60°C for 1 min. The studied genes were TBX21 (Hs00203436), GATA3 (Hs00203436), RORC (Hs01076122), STAT3 (Hs00374280), STAT4 (Hs00374280), STAT6 (Hs00598625) and FOXP3 (Hs01085834). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 5’-CCGCATCTTCTTGTGCAGTG-3’) was used as an endogenous control and mRNA were quantified using the ΔCt method [ΔCt = Ct (target gene)—Ct (endogenous gene)]. qPCR conditions were the same as described above for the gene expression analysis (Applied Biosystems).

dos Santos L.N., da Silva P.H., Alvim I.M., Nery J.A., Lara F.A., Sarno E.N, & Esquenazi D. (2016). Role of TEFFECTOR/MEMORY Cells, TBX21 Gene Expression and T-Cell Homing Receptor on Type 1 Reaction in Borderline Lepromatous Leprosy Patients. PLoS ONE, 11(10), e0164543.

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Quantification of RAC1, lncRNA-SOX2OT, and miR-194-5p

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For RAC1 gene quantification and lncRNA-SOX2OT and miR-194-5p expression analysis, total RNA was extracted using a TRIzol kit (Invitrogen). A NanoDrop ND-1000 instrument (NanoDrop) was used to measure the concentrations of the RNAs. A SuperScript® III RT-PCR kit (Life Technologies) was used to produce cDNA. The primers for real-time PCR were prepared as follows: RAC1: 5′-CCCCATTCTTGTTCAGATT-3′ (sense), 5′-TGCTTTACGCATCTGAGAACT-3′ (antisense); lncRNA-SOX2OT: 5′-GTTCATGGCCTGGACTCTCC-3′ (sense), 5′-ATTGCTAGCCCTCACACCTC-3′ (antisense); miR-194-5p: 5′-CCATGATT CCTTCATATTTGC-3′ (sense), 5′-GCAAATATGAAGGAATCATGG-3′ (antisense); GAPDH: 5′-GGTGGTCTCCTCTGACTTCAACA-3′ (sense) and 5′-CCAAATTCGTTGTCATACCAGGAAATG-3′ (antisense); and U6: 5′-CTCGCTTCGGCAGCACA-3′ (sense), 5′-AACGCTTCACGAATTTGCGT-3′ (antisense). The parameters for PCR amplification were as follows: 2 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 30 s at 60 °C. The real-time quantitative PCR data were analysed using the threshold cycle (Ct), and the relative gene expression levels were calculated by the 2^-△△Ct method.

Ni J., Zhang X., Li J., Zheng Z., Zhang J., Zhao W, & Liu L. (2021). Tumour-derived exosomal lncRNA-SOX2OT promotes bone metastasis of non-small cell lung cancer by targeting the miRNA-194-5p/RAC1 signalling axis in osteoclasts. Cell Death & Disease, 12(7), 662.

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Quantitative PCR Protocol for Mouse Genes

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qPCR was carried out using a Roche LightCycler 480 QPCR apparatus using intron-spanning qPCR primer pairs for mouse genes (Roche) (Supplemental Table 2). A SuperscriptIII RTPCR kit (Life Technologies) was used to generate template DNA from RNA. Reverse transcribed Superscript III product was used to generate PCR products with each primer pair. Product was used to generate standard QPCR curves. QPCR data were quantitated against murine Rpl13a run for each primer pair.

Ryan Z.C., Craig T.A., Salisbury J.L., Carpio L.R., McGee-Lawrence M., Westendorf J.J, & Kumar R. (2014). Enhanced Prostacyclin Formation and Wnt Signaling in Sclerostin Deficient Osteocytes and Bone. Biochemical and biophysical research communications, 448(1), 83-88.

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Isolation and Cloning of PAC1 Receptor Isoforms from Mouse Brain

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Total RNA was isolated from hippocampus, cortex and cerebellum of adult mice and rats, as well as from cultured rat hippocampal neurons (CHNs) using TRIzol reagent (Life Technologies). Obtained RNA was reverse transcribed into cDNA using SuperScript III RT-PCR kit (Life Technologies) as per manufacturer's instructions. Primer set [5′-ATGAATGACAGCACAGCTC-3′ (forward) and 5′-TCAGGTGGCCAAGTTGTCG-3′ (reverse)] spanning the PAC1 variable region in the third intracellular loop, based on known mouse and rat PAC1 sequences (NCBI reference sequences: NM_007407.4 and NM_133511.2, respectively), was used. Identification of various PAC1 isoforms expressed in mouse and rat brain regions was done based on differences in the isoform-specific molecular weights. PCR products of PAC1-Null, PAC1-Hop1 and PAC1-Hop2 isoforms were confirmed by sequencing. Subsequently, the primer set 5′-ATGGCCAGAACCCTGC-3″ (forward) and 5′-TCAGGTGGCCAAGTTGTCG-3′ (reverse), based on known mouse PAC1 sequence (NCBI reference sequences: NM_007407.4) was utilized for the amplification of full-length PAC1 receptor isoforms from mouse hippocampus cDNA. Individual full-length PAC1 isoforms were cloned in TOPO-TA cloning vector, and subsequently sub-cloned in pYFP-N1 plasmid (Clontech) for expressing mPAC1-YFP protein.

Gupte R.P., Kadunganattil S., Shepherd A.J., Merrill R., Planer W., Bruchas M.R., Strack S, & Mohapatra D.P. (2015). Convergent Phosphomodulation of the Major Neuronal Dendritic Potassium Channel Kv4.2 by Pituitary Adenylate Cyclase-activating Polypeptide. Neuropharmacology, 101, 291-308.

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Quantification of miR-761, ING4, and TIMP2 Expression

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Total RNA was isolated using Trizol reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's recommendations. The expression levels of miR-761 were quantified using the miRNAspecific TaqMan MiRNA Assay Kit (Applied Biosystems, Foster City, CA, USA), and normalized with U6 small nuclear RNA. Total RNA (1 μg) was subjected to reverse transcription with the SuperScript III RT PCR kit (Life Technologies). cDNA was applied to real-time PCR amplification with SYBR Green for ING4 and TIMP2 mRNA detection. β-actin was used as an internal control. The sequences for qRT-PCR primers were as follows:
ING4 sense primer (5'-CAAGGAATTTGGTGACGACAAG-3') and antisense primer (5'-TCCAGCCGCCGAATGT-3'); TIMP2 sense primer (5'-GTCATCTTGATCTCATAACGCTGG-3') and antisense primer (5'-AGCCCATCTGTACCTGTGGTTCA-3'); β-actin sense primer (5′-CAGGGAGTGATGGTGGGCA-3′) and antisense primer (5′-CAAACATCATCTGGTCATCTTCTC-3′).

Yan A., Yang C., Chen Z., Li C, & Cai L. (2015). MiR-761 Promotes Progression and Metastasis of Non-Small Cell Lung Cancer by Targeting ING4 and TIMP2. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 37(1).

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Quantifying Corneal Gene Expression

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Transgenic mouse corneas with and without injuries (n=6 per group) were collected at time intervals, placed in RLT buffer (Qiagen) and disrupted in MagNA Lyser Green Beads kit (Roche, Indianapolis, IN, US) using a MagNA Lyser Instrument (Roche). The lysate was processed with Qiashredder and RNeasy Miniprep (Qiagen, Germantown, MD, US) for total RNA extraction. RNA was precipitated by ethanol and quantified using NanoDrop One (Thermo Fisher). After reverse transcription using SuperScript III RT-PCR kit (Thermo Fisher) and random hexanucleotide primers, cDNA was assayed for target gene expression with specific target primers (Supplementary Table 1) using SYBR Green Real-Time Master Mix (Life Technologies, Carlsbad, CA, US) in a StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, US). Experiments were run in triplicate. The relative RNA abundance was assayed by 2^−ΔΔCT method after normalization with housekeeping glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and 18S genes and fold changes were expressed as mean ± standard deviation (SD).

Khandaker I., Funderburgh J.L., Geary M.L., Funderburgh M.L., Jhanji V., Du Y, & Yam G.H. (2020). A novel transgenic mouse model for corneal scar visualization. Experimental eye research, 200, 108270.

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Corneal Endothelium and Trabecular Tissue Analysis

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From human cornea (n = 11), central CE was collected after DM peeling (central 8 mm diameter). The posterior limbus was collected by an inward peeling method (https://www.youtube.com/watch?v=wQlPhHCJgmM&t=121s). Under stereo-microscope, the tissue was further dissected to separate the innermost transparent PE with <0.5 mm width, the outermost pigmented TM tissue, and the remaining intermediate non-pigmented smooth TZ. All samples were washed at least three times in ice-cold PBS, followed by preservation in Trizol reagent (Sigma-Aldrich). Total RNA was extracted using RNeasy kit (Qiagen, Hilden, Germany) and on-column RNase-free DNase kit (Qiagen). After reverse transcription using Superscript III RT-PCR kit (ThermoFisher), cDNA was assayed for candidate gene expression with specific primer pairs (Supplementary Table S3) by quantitative real-time PCR (qPCR) using Sybr Green Supermix (BioRad, Herculus, CA, USA) or Taqman assay in GFX96 real-time system (BioRad). Experiments were run in quadruplicate. The relative gene expression (ΔCT) normalized with mean CT of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (CT_GAPDH) or β-actin (ACTB) (CT_ACTB) was obtained, and fold change to the central CE was expressed as mean ± SD.

Yam G.H., Seah X., Yusoff N.Z., Setiawan M., Wahlig S., Htoon H.M., Peh G.S., Kocaba V, & Mehta J.S. (2019). Characterization of Human Transition Zone Reveals a Putative Progenitor-Enriched Niche of Corneal Endothelium. Cells, 8(10), 1244.

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Quantitative PCR for Gene Expression

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Total RNA from cells was extracted using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.). cDNA was produced by reverse transcription using the SuperScript^® III RT-PCR kit according to the manufacturer’s instructions (Thermo Fisher Scientific, Inc.). The amplification of fluorescence signals was detected by a fluorescence thermal cycler (Bio-Rad Laboratories, Inc., United States). RT-qPCR was performed (Novoprotein, Shanghai, China) using the following primers: RPS3 sense, 5′-GCGAGTTACACCAACCAGGA-3′ and antisense, 5′-ATGAACCGCAGCACACCATA-3′; β-actin sense, 5′-AGCAGCATCGCCCCAAAGTT-3′ and antisense, 5′-GGGCACGAAGGCTCATCATT-3′. B -Actin was set as an internal control for cellular mRNAs. The parameters for PCR quantification were as follows: 2 min at 95°C, followed by 40 cycles of 15 s at 95°C and 30 s at 60°C. The results of qPCR were defined using the quantification cycle (Cq), and 2^–ΔΔ^C^q was used to calculate the relative expression levels (Livak and Schmittgen, 2001 (link)).

Sun M.Y., Xu B., Wu Q.X., Chen W.L., Cai S., Zhang H, & Tang Q.F. (2021). Cisplatin-Resistant Gastric Cancer Cells Promote the Chemoresistance of Cisplatin-Sensitive Cells via the Exosomal RPS3-Mediated PI3K-Akt-Cofilin-1 Signaling Axis. Frontiers in Cell and Developmental Biology, 9, 618899.

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qRT-PCR Analysis of Corneal Gene Expression

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Corneas were isolated and placed in RLT reagent, and disrupted with MagNA Lyser green beads in a MagNA Lyser Instrument (Roche). The lysates were processed using QiaShredder (Qiagen), and total RNA was extracted using RNeasy Mini kit (Qiagen) with on-column RNase-free DNase digestion (Qiagen), per manufacturer’s instruction. RNA was quantified using NanoDrop One (Thermo Fisher) and 500 ng RNA was reverse-transcribed to cDNA using SuperScript III RT-PCR kit (Thermo Fisher) and random hexanucleotide primers. Target gene expression was performed with specific primers (Table S2) using SYBR Green Real-Time Master Mix (Life Technologies, Carlsbad, CA) in a QuantStudio 3 Real-Time PCR System (Applied Biosystems). Experiments were done in triplicate and relative RNA abundance was assayed by the 2^-ΔΔCt method after normalization with housekeeping 18S gene and fold changes were expressed as mean ± SD. Significance was determined by non-parametric Mann-Whitney U test. Primer validation by agarose gel electrophoresis and melting curves is shown in Supplementary Fig. S9.

Yam G.H., Yang T., Geary M.L., Santra M., Funderburgh M., Rubin E., Du Y., Sahel J.A., Jhanji V, & Funderburgh J.L. (2022). Human corneal stromal stem cells express anti-fibrotic microRNA-29a and 381-5p – A robust cell selection tool for stem cell therapy of corneal scarring. Journal of Advanced Research, 45, 141-155.

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