Rt2 first strand kit

Manufactured by Qiagen

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The RT2 First Strand Kit is a laboratory reagent used for the reverse transcription of RNA to cDNA. It provides the necessary components for the conversion of RNA to cDNA, which is a crucial step in various molecular biology applications, such as gene expression analysis and real-time PCR.

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Lab products found in correlation

1 647 protocols using rt2 first strand kit

Senescence Markers and Sirt1 Expression in Brain Microvessels

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Total RNA from the human and mouse brain microvessels was prepared using TRIZOL (Thermo Fisher Scientific). Single strand cDNA was synthesized using RT² first strand kit (Qiagene). Analysis of senescence marker mRNA expression in human and mouse brain microvessels was done using Human and Mouse Aging PCR-array (Qiagen), according to the manufacturer’s protocol. The mRNA expression pattern was evaluated using software provided by the manufacturer.
Analysis of Sirt1 mRNA expression in microvessels isolated from human and mouse brain tissue as well as in mBMEC, was performed using TaqMan real-time PCR assay on Applied Biosystem 7500 Real time PCR system (Applied Biosystems). Single strand cDNA was synthesized using RT² first strand kit (Qiagen). Real-Time PCR was performed for SirT1 using commercially available primers (Qiagen). β-actin was used to normalize the amount of SirT1 mRNA in each sample.

Stamatovic S.M., Martinez G.R., Hu A., Choi J., Keep R.F, & Andjelkovic A.V. (2018). Decline in Sirtuin-1 expression and activity plays a critical role in blood-brain barrier permeability in aging. Neurobiology of disease, 126, 105-116.

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Gene Expression Analysis of DENV-Infected Cells

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Human Antiviral Response RT² Profiler PCR Array and Human Cytokines and Chemokines RT² Profiler PCR Array (QIAGEN, Hilden Germany) were selected for analysis of gene expression in response to DENV infection and cytokine/chemokine production, respectively. Total RNA was isolated either from mock-infected HepG2 cells as control cells or from DENV-2-infected cells in the presence or absence of 20 μM of α-MG using an RT² First Strand Kit (QIAGEN). For RNA quality control, the RNA concentration and ribosomal RNA band integrity of each sample were measured by Qubit 2.0 (Invitrogen) and an Agilent Bioanalyzer 2100 (Agilent Technologies), respectively. One microgram (μg) of RNA sample was transcribed into cDNA using an RT² First Strand Kit (QIAGEN). The cDNA was further mixed with SYBR Green RT² qPCR Mastermix (QIAGEN), and equal volumes were aliquoted into the wells of the PCR arrays containing 84 antiviral response-related genes, and 84 cytokine and chemokine genes. The PCR amplification steps were performed in a Roche LightCycler 480 instrument (Roche Diagnostics) and the Ct values were analyzed using the web-based program http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php for 2^−ΔΔCt analysis. The results are presented as fold increase or decrease relative to the results of mock-infected HepG2 cells.

Tarasuk M., Songprakhon P., Chieochansin T., Choomee K., Na-Bangchang K, & Yenchitsomanus P.T. (2022). Alpha-mangostin inhibits viral replication and suppresses nuclear factor kappa B (NF-κB)-mediated inflammation in dengue virus infection. Scientific Reports, 12, 16088.

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Fibrosis-related gene expression analysis

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To analyze the expression of genes related to fibrosis, we purified RNA from the expanded cells by using ISOGEN II (NIPPON GENE). The concentration of RNA was determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific), and 1 μg of RNA was used to generate cDNA using the RT² First Strand Kit (SABiosciences, a Qiagen Company). The mouse fibrosis RT² Profiler PCR array was performed according to the manufacturer's instructions (#330231 PAMM-120ZA, SABiosciences). This PCR array profiles 84 key genes involved in dysregulated tissue remodeling during the repair and healing of wounds. The fold change of expression was calculated using the SABiosciences web-based data analysis program. The results represent the mean expression of three independent samples.

Peng Y.H., Xiao J., Yan C., Luo L, & Li T.S. (2019). Potential Role of the Resident Mesenchymal Stem-Like Cells in Renal Fibrogenesis after Ureteral Obstruction. Stem Cells International, 2019, 2543171.

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Pathway-Focused Analysis of Liver Regeneration

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To search the factors associated with liver regeneration, a pathway-focused array analysis was performed. Briefly, total RNA was isolated from half of the freshly collected liver tissues from each mouse by using an RNeasy Mini Kit (Qiagen). We used 1 mg of total RNA from each sample and pooled all of the 4 RNA samples at the same time point in each group to generate cDNA using the RT2 First Strand Kit (SABiosciences). A mouse extracellular matrix and adhesion molecule PCR array and a cytokine and chemokine PCR array, each containing a total of 84 genes, were used according to the manufacturer’s instructions (SABiosciences)28 (link)29 (link)30 (link). All of the data were normalized to the expression of housekeeping genes, and the expression level for each gene was quantified based on the cycle threshold (Ct). Data analysis was performed with a web-based analysis program (SABiosciences, http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php). We selected the genes that were up- and down-regulated by more than 1.5-fold for further categorization analysis according to their biological function.

Zhang S., Li T.S., Soyama A., Tanaka T., Yan C., Sakai Y., Hidaka M., Kinoshita A., Natsuda K., Fujii M., Kugiyama T., Baimakhanov Z., Kuroki T., Gu W, & Eguchi S. (2016). Up-regulated extracellular matrix components and inflammatory chemokines may impair the regeneration of cholestatic liver. Scientific Reports, 6, 26540.

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Quantifying Neurotransmitter Receptors in iPSC-Derived Neurons

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For neurotransmitter receptors analysis, iPSC-derived neurons from ASD and related controls were rapidly frozen and placed in TRIzol (Life Technologies) for subsequent RNA isolation and purification. One microgram of RNA of each individual/clone were converted into cDNA using the RT² First Strand Kit (SABiosciences). Quantitative RNA expression analysis of 84 neurotransmitter receptors-related genes was simultaneously analyzed using human RT² Profiler PCR Array (#PAHS-060Z; SABiosciences) on a Bio-Rad CFX96 following the manufacturer’s instructions. Data normalization was based on correcting all Ct values for the average Ct values of five constantly expressed housekeeping genes present on the array, and reported as fold changes over similar values from controls samples.

Marchetto M.C., Belinson H., Tian Y., Freitas B.C., Fu C., Vadodaria K., Beltrao-Braga P., Trujillo C.A., Mendes A.P., Padmanabhan K., Nunez Y., Ou J., Ghosh H., Wright R., Brennand K., Pierce K., Eichenfield L., Pramparo T., Eyler L., Barnes C.C., Courchesne E., Geschwind D.H., Gage F.H., Wynshaw-Boris A, & Muotri A.R. (2016). Altered proliferation and networks in neural cells derived from idiopathic autistic individuals. Molecular psychiatry, 22(6), 820-835.

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Profiling Mouse WNT Signaling Targets

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The Mouse WNT Signalling Targets (PAMM-243Z SABiosciences) was utilised to investigate a panel of 84 WNT specific genes in mouse intestinal tissue as per manufacturer’s instructions. Briefly, 500 ng of RNA was converted to cDNA using RT² First Strand Kit (SABiosciences). The resultant cDNA product was immediately amplified by qPCR using RT² SYBR Green qPCR Master Mix (SABiosciences) and a Roche Lightcycler 480. The Ct values (threshold cycle) for both KI/KI and WT/WT samples were evaluated using the provided web-based portal (http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php) and normalised to housekeeping genes present on the array. This analysis generated fold-change, which represents the normalized gene expression in the KI/KI samples compared to the normalized gene expression in the WT/WT samples. Following the portal guidelines, a significant difference in expression was set at greater than or less than 3-fold difference in fold-change.

Hey F., Giblett S., Forrest S., Herbert C, & Pritchard C. (2016). Phosphorylations of Serines 21/9 in Glycogen Synthase Kinase 3α/β Are Not Required for Cell Lineage Commitment or WNT Signaling in the Normal Mouse Intestine. PLoS ONE, 11(6), e0156877.

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Hypoxia-Reoxygenation Stress Response

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Messenger RNAs of H/R-adapted and control PT cultures (normoxic and after hypoxia (48 h)/reoxygenation (24 h)) were isolated (Qiagen RNA extraction kit, Hilden, Germany) and reversely transcribed in cDNA (RT² First Strand Kit, SABiosciences, Qiagen). mRNA fragments were amplified by the use of an “oxidative stress” and an “apoptosis” PCR array (RT Profiler PCR Array, SABiosciences, Qiagen, Hilden, Germany) and a Roche Light Cycler 480. C_t (threshold cycle) values of the PCR amplifications were normalized to that of the housekeeper genes. Mouse UCP-3 and GAPDH specific cDNAs were amplified by QuantiTect Primer Assays (#QT00115339 and QT01658692, respectively, Qiagen).

Braun N., Klumpp D., Hennenlotter J., Bedke J., Duranton C., Bleif M, & Huber S.M. (2015). UCP-3 uncoupling protein confers hypoxia resistance to renal epithelial cells and is upregulated in renal cell carcinoma. Scientific Reports, 5, 13450.

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qRT-PCR analysis of cell junctions

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Control and ShAkt1 cells were used for the qRT-PCR arrays. Briefly, cells were lysed and RNA was isolated using RNAase Mini plus Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Next, cDNA was generated by RT2 First Strand Kit (SABiosciences, Frederick, MD), mixed with qPCR SyberGreen master mix and loaded into Tight-junction and Adherens-junction RT2 Profiler PCR Array plates. Reading was completed in Eppendorf Mastercycler realplex-2 equipment (Hauppauge, NY).

Gao F., Artham S., Sabbineni H., Al-Azayzih A., Peng X.D., Hay N., Adams R.H., Byzova T.V, & Somanath P.R. (2016). Akt1 promotes stimuli-induced endothelial-barrier protection through FoxO-mediated tight-junction protein turnover. Cellular and molecular life sciences : CMLS, 73(20), 3917-3933.

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Comprehensive RNA Extraction and cDNA Synthesis

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Total RNA was extracted using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and purified with the RNeasy MinElute Cleanup kit (Qiagen GmbH) according to the manufacturer's instructions. DNase enzyme digestion (Qiagen GmbH), was performed to exclude genomic DNA contamination. Purified RNA was eluted with 14 µl of RNase-free water (Qiagen GmbH). A total of 500 ng of purified RNA were reverse transcribed into cDNA using the RT² First Strand kit (SABiosciences Corp.) in a volume of 20 µl reaction and diluted with 91 µl of nuclease-free water, following the manufacturer's instructions.

Zamagni A., Pasini A., Pirini F., Ravaioli S., Giordano E., Tesei A., Calistri D., Ulivi P., Fabbri F., Foca F., Delmonte A, & Molinari C. (2020). CDKN1A upregulation and cisplatin-pemetrexed resistance in non-small cell lung cancer cells. International Journal of Oncology, 56(6), 1574-1584.

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Immune Response Gene Expression Profiling

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Total cellular mRNA was purified using the RNeasy kit (Qiagen, Hilden, Germany). Reverse-transcription of total mRNA was performed using the RT² First Strand Kit (SABiosciences, Hilden, Germany). The expression of 84 genes involved in the human innate and adaptive immune responses was evaluated using the RT² profiler™ PCR Array (SABiosciences) according to the manufacturer’s recommendations. The ΔΔCt method was applied to calculate the fold changes in gene expression for each gene relative to uninfected control cells using the web-based RT2 profiler PCR Array Data Analysis software (SABiosciences).

Hoffmann J., Machado D., Terrier O., Pouzol S., Messaoudi M., Basualdo W., Espínola E.E., Guillen R.M., Rosa-Calatrava M., Picot V., Bénet T., Endtz H., Russomando G, & Paranhos-Baccalà G. (2016). Viral and bacterial co-infection in severe pneumonia triggers innate immune responses and specifically enhances IP-10: a translational study. Scientific Reports, 6, 38532.

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