RNA was isolated using an RNA Miniprep Kit (Sigma, St. Louis, MO, USA). Next, DNase treatment was performed, and 1 µg RNA was used to prepare cDNA. LTβ, A20, CEACAM1, CEACAM5, CEACAM6, CCL2, CXCL10, and GAPDH primer sequences are shown in Table 1 . Relative mRNA levels were calculated using the equation 2−∆ct after normalizing values to the housekeeping gene GAPDH. Fold mRNA expression was calculated using the 2−∆∆ct formula. Thus, values were first normalized to the housekeeping gene and afterwards to the values of the control samples.
Rna miniprep kit
Manufactured by Merck Group
Sourced in United States
The RNA miniprep kit is a laboratory equipment designed to extract and purify RNA from small biological samples. It provides a simple and efficient method to isolate high-quality RNA for various downstream applications, such as reverse transcription and gene expression analysis.
Automatically generated - may contain errors
Lab products found in correlation
Superscript 3 first strand cdna synthesis system, by Thermo Fisher Scientific (2 mentions)
Cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Abi prism 7900ht sequence detection system, by Thermo Fisher Scientific (1 mentions)
Maxima first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Steponeplus machine, by Thermo Fisher Scientific (1 mentions)
Fast sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Random primer, by Thermo Fisher Scientific (1 mentions)
M mlv, by Thermo Fisher Scientific (1 mentions)
Quantistudio 3 pcr system, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
7 protocols using rna miniprep kit
1
Gene Expression Analysis by RT-qPCR
2
Total RNA Isolation and RT-PCR Analysis
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Total RNA was isolated using an RNA miniprep kit (Sigma Santa Clara, CA, USA). For RT-PCR, the cDNA was synthesized using a cDNA reverse transcription kit (Applied Biosystems, Zurich, Switzerland), and the PCR kits were purchased from Sigma. Based on the LRIG1, BCL-2, and β-actin gene sequences, three pairs of gene-specific primers were designed: LRIG1 sense 5'-ttgctgatgttgtttcgctg-3'and antisense 5'-tgatggtctgtcacggtcg-3', BCL-2 sense 5'-ttctttgagttc ggtggggtc-3' and antisense 5'-tgcatatttgtttggggcagg-3', β-actin sense 5'-gtccaccgcaaatgcttcta-3' and antisense 5'-tgctgtcaccttcaccgttc-3'.
3
Broad-spectrum Viral Screening Protocol
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All samples were screened for five priority viral families using a conventional PCR approach. RNA was extracted from samples with the RNA MiniPrep Kit (Sigma-Aldrich) and cDNA transcribed with the SuperScript III First Strand cDNA Synthesis System (Invitrogen). Broadly reactive PCR assays were then used to screen all samples for Coronaviridae, Filoviridae, Flaviviridae, Orthomyxoviridae, and Paramyxoviridae, following protocols outlined in Lee et al. (24 (link)) and Huong et al. (25 (link)). Assays developed by Watanabe et al. (26 (link)) and Quan et al. (27 (link)) [with a modification, as described in Lee et al. (24 (link))] targeting two regions of the RNA-dependent RNA polymerase (RdRp) gene were used to screen samples for CoVs with consensus nested-PCRs. In each test batch, controls included distilled water (negative control) and a synthetic plasmid primer with binding sites for each assay (positive control).
4
Detecting Coronavirus via Nested-PCR
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RNA was extracted (RNA MiniPrep Kit, Sigma-Aldrich) and cDNA transcribed (SuperScript III First Strand cDNA Synthesis System, Invitrogen). Coronavirus RNA was detected using two broadly reactive consensus nested-PCR assays targeting the RNA dependent RNA polymerase (RdRp) gene [28 (link),29 (link)]. The positive control was a synthetic plasmid containing the primer-binding sites for both assays. Distilled water was used as a negative control and included in each test batch. PCR products were visualized using 1.5% agarose gels, and bands of the correct size were excised, cloned, and sequenced by Sanger dideoxy sequencing using the same primers as for amplification.
5
Quantitative Real-Time PCR Analysis
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As the proliferation assay demonstrated that 1 ng/µL CpdA reduced the proliferation to 76%, this concentration was selected for mRNA expression analysis. After treatment, total mRNA was extracted using the RNA Miniprep Kit (Sigma-Aldrich, Taufkirchen, Germany), according to the manufacturer’s protocol. First strand cDNA was synthesized using 1 µg of total RNA (DNase-treated) and the Maxima first strand cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA) and stored at −20 °C until use. All real-time PCRs were performed according to a standard protocol as described previously [39 (link),56 (link)]. Quantitative real-time PCR was performed using the ABI PRISM® 7900HT sequence detection system from Applied Biosystems (Foster City, CA, USA). The fluorescence threshold value was calculated using ABI PRISM® 7900HT sequence detection system software (version 2.2); the housekeeping gene β-actin was used for normalization. The primers used for real-time PCR are listed in Table 1 .
6
Transcript Quantification by RT-MLPA and qPCR
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Total ribonucleic acid (RNA) for reverse transcriptase multiplex ligation-dependent probe amplification (RT-MLPA) or quantitative real time PCR was extracted using an RNA miniprep kit (Sigma, St. Louis, MO, USA). RT-MLPA procedure was performed as previously described.42 (link) qPCR was performed with Fast SYBR Green Master Mix on a StepOne Plus machine (Life Technologies, Breda, The Netherlands) using the following primers: Hprt forward 5′-CCTGGCGTCGTGATTAGTGA-3′, Hprt reverse 5′-CGAGCAAGACGTTCAGTCCT-3′, MCL1 forward 5′-TCGTAAGGACAAAACGGGAC-3′, MCL1 reverse 5′-CATTCCTGATGCCACCTTCT-3′, BCL2 forward 5′-ATGTGTGTGGAGAGCGTCAA-3′, BCL2 reverse 5′-CAGTTCCACAAAGGCATCCCAG-3′, BCLX forward 5′-GTATTGGTGAGTCGGATCGC-3′, BCLX reverse 5′-TGCTGCATTGTTCCCATAGA-3′, BFL1 forward 5′-TCATATTTTGTTGCGGAGTTCA-3′, BFL1 reverse 5′-TCCAGCCAGATTTAGGTTCAA-3′, HUWE1 forward 5′-TAATCATGCCGCAGAAGAAG-3′, HUWE1 reverse 5′-CAACCTGCTGATTAGGCTCA-3′, FBXW7 forward 5′-ACCAACTCTCCTCCCCATTC-3′, FBXW7 reverse 5′-TTTCCCAAAGAAAAAGAGCG-3′, BTRC forward 5′-TGCAAGAGAAGGCACTCAAG-3′, BTRC reverse 5′-CCTGGGTTATACAGGCATCG-3′. The results were normalized to expression of Hprt using the ΔΔCt method.34 (link)
7
Quantitative RT-PCR for Gene Expression
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