High capacity cdna reverse transcription kit

Manufactured by Qiagen

Sourced in Germany, United States

The High-Capacity cDNA Reverse Transcription Kit is a laboratory tool designed for the efficient conversion of RNA into complementary DNA (cDNA). The kit provides the necessary components, including reverse transcriptase enzyme, buffer, and random primers, to facilitate the reverse transcription process.

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Reverse transcription kit

40 protocols using high capacity cdna reverse transcription kit

Quantification of Mitochondrial DNA and Gene Expression

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Relative mitochondrial DNA copy number was analyzed by absolute QPCR using TaqMan probes. Mitochondrial and nuclear DNA were detected by using ND2 and Rplp0 single tube Taqman real-time PCR assay, respectively (cat. no. 4331182; Life Technologies, Austin, TX, USA).
RNA was extracted by RNeasy kit (cat. no. 74106; Qiagen, Hilden, Germany) and cDNA synthesis was carried out by the high capacity cDNA reverse transcription kit (cat. no. 4368813; Life Technologies). Gene expression analysis was carried out using single tube Taqman real-time PCR assays (cat. no. 4331182; Life Technologies).

Fortini P., Ferretti C., Iorio E., Cagnin M., Garribba L., Pietraforte D., Falchi M., Pascucci B., Baccarini S., Morani F., Phadngam S., De Luca G., Isidoro C, & Dogliotti E. (2016). The fine tuning of metabolism, autophagy and differentiation during in vitro myogenesis. Cell Death & Disease, 7(3), e2168-.

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EphA2 Expression Quantification by RT-qPCR

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TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used for total RNA extraction from all types of cells according to standard procedures. The detection of EphA2 was performed via RT-qPCR using a high-capacity cDNA Reverse Transcription kit (Qiagen AB) and SYBR Premix Ex Taq (Qiagen AB). The conditions for cDNA synthesis were as follows: 42°C for 30 min and 85°C for 5 sec. The thermocycling conditions were as follows: 95°C for 3 min; 39 cycles of 95°C for 5 sec, 56°C for 10 sec, 72°C for 25 sec; 65°C for 5 sec; 95°C for 50 sec. The relative expression levels were normalized to endogenous control GAPDH and were expressed as 2^−ΔΔCq (22 (link)). The sequences of the primers were as follows: EphA2 forward, 5′-CTGGTCTGCAAGGTGTCTGA-3′ and reverse, 5′-TTGGACAACTCCCAGTAGGG-3′; and GADPH forward, 5-GATATTGTTGCCATCAATGAC-3 and reverse 5-TTGATTTTGGAGGGATCTCG-3.

Li X., Li D, & Ma R. (2022). ALW-II-41-27, an EphA2 inhibitor, inhibits proliferation, migration and invasion of cervical cancer cells via inhibition of the RhoA/ROCK pathway. Oncology Letters, 23(4), 129.

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Quantifying mRNA Expression After Knockdown

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The RNeasy Mini Kit was used to isolate mRNA after transfection according to manufacturer’s instructions. Reverse transcription was performed with the High-Capacity cDNA Reverse Transcription Kit (all Qiagen, Venlo, The Netherlands). Finally, a Rotor-Gene Q machine was used for the chain reaction. Taq Man probes (all Applied Biosystems, Foster City, CA, USA) normalizing to 18S expression were used (Supplementary Table S2). RNA quality was determined via biophotometer (Eppendorf, Hamburg, Germany) with A260/280 rates between 1.8 and 2.0 considered appropriate, as before (Troschel et al. 2018 (link)). Data are expressed as fold changes using the 2^-∆∆Ct method comparing MSI-1 knockdown cells to controls.

Troschel F.M., Palenta H., Borrmann K., Heshe K., Hua S.H., Yip G.W., Kiesel L., Eich H.T., Götte M, & Greve B. (2021). Knockdown of the prognostic cancer stem cell marker Musashi-1 decreases radio-resistance while enhancing apoptosis in hormone receptor-positive breast cancer cells via p21WAF1/CIP1. Journal of Cancer Research and Clinical Oncology, 147(11), 3299-3312.

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Quantifying Neuroinflammatory Gene Expression

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On day 7, a sub-set of animals (N=6 per group) was sacrificed by conscious decapitation and blood collection and cryostat tissue punches were taken from the PVN of fresh frozen brains (Kapusta et al., 2012 (link); Kapusta et al., 2013 (link)). Total RNA was isolated using RNeasy kits (Qiagen) according to the manufacturer’s instructions, and 200 ng of purified RNA was reverse transcribed with a high-capacity cDNA reverse transcription kit (Qiagen). The IL-1β, IL-6, tumor necrosis factor-α, and IL-10 mRNA levels were analyzed by quantitative real-time PCR using specific primers and probes in a PRISM 7000 sequence detection system (Applied Biosystems) (Shi et al., 2010 (link)). Data were normalized to 18s ribosomal RNA, and the 2^−ΔΔCT method was used to calculate relative changes in gene expression (Zuriaga et al., 2017 ).

Moreira J.D., Chaudhary P., Frame A.A., Puleo F., Nist K.M., Abkin E.A., Moore T.L., George J.C, & Wainford R.D. (2019). Inhibition of microglial activation in rats attenuates paraventricular nucleus inflammation in Gαi2 protein‐dependent, salt‐sensitive hypertension. Experimental Physiology, 104(12), 1892-1910.

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Quantitative RT-PCR Analysis of Immune Genes

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RNA was isolated from uninfected and infected dNK cells using TRIzol and chloroform and then precipitated using isopropanol. Generation of cDNA was accomplished using a Thermo Fisher High-Capacity cDNA Reverse Transcription Kit following the manufacturer’s instructions, and RT-PCR was catalyzed using an RT-PCR synthesis kit (Qiagen, Germany). The following primers were used: h2B4-A: F: 5′-CCTCTACTGCCTGGAGGTCACCAG-3′, R: 5′-CCACTTGGCATCTCCCTCTGTCC-3′; h2B4-B: F: 5′-GCCTTCCAATACTTCC-3′, R: 5’-TGGAAGCAGAGATTC-3′; SHP-2: F: 5′-AAAGGGGAGAGCAATGACGG-3′, R: 5′-GGGGCTGCTTGAGTTGTAGT-3′. Measurements were normalized to actin: F: 5′-GCACCGTCAAGGCTGAGAAC-3′, R: 5′-TGGTGAAGACGCCAGTGGA-3′.

Xu X., Zheng G., Ren Y., He X., Peng B., Hu X, & Liu W. (2022). A novel 2B4 receptor leads to worse pregnancy outcomes by facilitating TNF-α and IFN-γ production in dNK cells during Toxoplasma gondii infection. Parasites & Vectors, 15, 337.

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Exosomal RNA Extraction and Analysis

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Whole-RNA extracts were prepared using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and total RNA from exosomes was isolated with the exoRNeasy Midi Kit (Qiagen, Valencia, CA, USA) according to the standard procedure. Then extracted RNA was interacted with Rnase R (Epicentre, Madison, WI, USA), followed by incubation with RNeasy MinElute Cleanup Kit (Qiagen). RNA samples were reversely transcribed into cDNA using a High Capacity cDNA Reverse Transcription Kit (Qiagen), and cDNA amplification was carried out with SYBR Premix Ex Taq (Qiagen). Glyceraldehyde 3-phosphate dehydrogenase (GADPH) and U6 small nuclear B noncoding RNA (U6) were used as internal references to normalize the fold changes using 2^−ΔΔCt method. The specific primer sequences were listed as follows:

Hu K., Liu X., Li Y., Li Q., Xu Y., Zeng W., Zhong G, & Yu C. (2020). Exosomes Mediated Transfer of Circ_UBE2D2 Enhances the Resistance of Breast Cancer to Tamoxifen by Binding to MiR-200a-3p. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 26, e922253-1-e922253-12.

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RNA Extraction and qRT-PCR Analysis

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The QIAGEN RNeasy Kit and High Capacity cDNA Reverse Transcription Kit were used to extract total RNA from the verified samples and conduct reverse transcription reactions. qRT-PCR was performed in a volume of 20 µl with Power SYBR™ Green PCR Master Mix on a QuantStudio 3 and 5 System (Applied Biosystems). GAPDH was used as a housekeeping control. The sequences of the primers are shown in Table 2.

Wu J., Guo J., Fang Q., Liu Y., Li C., Xie W, & Zhang Y. (2023). Identification of biomarkers associated with the invasion of nonfunctional pituitary neuroendocrine tumors based on the immune microenvironment. Frontiers in Endocrinology, 14, 1131693.

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RNA Isolation and Gene Expression Quantification

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RNA was isolated from 30 mg of tissue (lung, liver, spleen, and kidney) using RNeasy mini kit (Cat. No. 74106, Qiagen) and was converted into cDNA using a high‐capacity cDNA reverse transcription kit (Cat. No. 4368813, Applied Biosystems) following manufacturer's instructions. Gene expression was then quantified using gene‐specific primers (Table S1) and fast SYBR green master‐mix (Cat. No. 4385617, Applied Biosystems) as per the manufacturer's instructions. GAPDH was used to normalize the expression levels across all samples and the gene expression level in untreated tissues was used as baseline to compare the fold change in expression between groups. Fold change in gene expression was determined using ΔΔCT method.

Budamagunta V., Kumar A., Rani A., Bean L., Manohar‐Sindhu S., Yang Y., Zhou D, & Foster T.C. (2023). Effect of peripheral cellular senescence on brain aging and cognitive decline. Aging Cell, 22(5), e13817.

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RNA Extraction and Gene Expression Analysis

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RNA isolated from cells using RNeasy mini kit (Cat. No. 74106, Qiagen, Hilden, Germany) was converted into cDNA using a high capacity cDNA reverse transcription kit (Cat. No. 4368813, Applied Biosystems, Foster City, CA, USA). Gene expression was then quantified using gene specific primers (Supplementary Table 2) and fast SYBR green master-mix (Cat. No. 4385617, Applied Biosystems. Foster City, CA, USA) as per the manufacturer’s instructions. The expression of GAPDH was used for normalization. Level of gene expression in untreated NC cells was used as baseline and fold change in gene expression was defined based on ΔΔCT method.

Budamagunta V., Manohar-Sindhu S., Yang Y., He Y., Traktuev D.O., Foster T.C, & Zhou D. (2021). Senescence-associated hyper-activation to inflammatory stimuli in vitro. Aging (Albany NY), 13(15), 19088-19107.

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Comprehensive RNA Isolation and Expression Analysis

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Total RNA was isolated using RNeasy mini kit (Cat. No. 74106, Qiagen, Valencia, CA), with on-column digestion with DNase I digestion (Cat. No. 79254, Qiagen). Total RNA (0.2–1 μg) was used to synthesize cDNA using the Prime Script^TM RT Master Mix (Cat. No. RR036A, Takara, Mountain View, CA) for coding genes, QuantiTect Reverse Transcription Kit (Qiagen) or High-Capacity cDNA Reverse Transcription kit (Cat. No. 4368814, Thermo Fisher Scientific) for lncRNAs, and qScript microRNA cDNA Synthesis Kit (Quanta Bio, Beverly, MA) for miRNAs. Gene expression was analyzed by quantitative PCR (qPCR) using SYBR Green reagent and TaqMan assays (Cat. No. 4367659 and 7352042 respectively, Applied Biosystems, Foster City, CA) with gene specific primers (Online only Supplementary Table II), and PerfeCTa SYBR® Green SuperMix (Quantabio) for miRNAs in in triplicate on 7500 Fast Real-Time PCR system (Applied Biosystems). Relative gene expression between control and treated groups were determined using 2^−ΔΔCt method^{24 (link), 25 (link)} after normalization with Ppia, 18S, HPRT1 and Rplp0 (for coding genes and lncRNAs) and U6 (for miRNAs).

Das S., Reddy M.A., Senapati P., Stapleton K., Lanting L., Wang M., Amaram V., Ganguly R., Zhang L., Devaraj S., Schones D.E, & Natarajan R. (2018). Diabetes-induced lncRNA Dnm3os regulates macrophage functions and inflammation via nuclear mechanisms. Arteriosclerosis, thrombosis, and vascular biology, 38(8), 1806-1820.

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