Total RNA was extracted from the RABV strains (ERAGS and ERA) using an RNA extraction kit (Bioneer, Korea) in accordance with the manufacturer's instructions. The extracted RNA was eluted in 50 μL RNase- and DNase-free water and subjected to RT-PCR. Multiplex RT-PCR was performed using OneStep RT-PCR reagent (Qiagen, Germany). The RT-PCR mixtures contained 2 µL denatured RNA, 1 µL each of the four primers (10 pmol), 5 µL 5× buffer (12.5 mM MgCl2), 1 µL dNTP mix, 1 µL enzyme mix (reverse transcriptase and Taq polymerase), and 12 µL distilled water. The PCR profile consisted of cDNA synthesis at 50°C for 30 min, followed by 40 cycles of 95°C for 30 sec, 55°C for 30 sec, and 72°C for 30 sec, with a final extension at 72°C for 5 min. The PCR products were electrophoresed on a 2.0% agarose gel for 30 min and visualized using Redsafe™ Nucleic acid staining solution (iNtRON Biotechnology, Korea).
Rna extraction kit
Manufactured by Bioneer
Sourced in Cameroon
The RNA extraction kit is a laboratory tool designed to isolate and purify ribonucleic acid (RNA) from a variety of biological samples. This kit utilizes a chemical-based extraction method to effectively separate RNA from other cellular components, allowing for its subsequent analysis and study.
Automatically generated - may contain errors
Lab products found in correlation
Redsafe nucleic acid staining solution, by iNtRON Biotechnology (2 mentions)
Accupower rocketscript cycle rt premix dt20, by Bioneer (2 mentions)
Ssofast evagreen supermix, by Bio-Rad (2 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (2 mentions)
Dna extraction kit, by Bioneer (2 mentions)
Pgem t easy vector system, by Promega (1 mentions)
Onestep qrt pcr master mix, by Biofact (1 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (1 mentions)
Real time pcr system, by Thermo Fisher Scientific (1 mentions)
Sybr green, by Toyobo (1 mentions)
9 protocols using rna extraction kit
1
RABV RNA Extraction and RT-PCR Analysis
2
GFP Gene Amplification and Sequencing
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RNA from ERAGS and ERAGS-GFP was extracted using an RNA Extraction Kit (Bioneer, Korea) according to the manufacturer's instructions. A 1 step RT-PCR Premix Kit (Bioneer) was used to amplify the GFP gene with the primer listed in Table 1 . The reaction mixture (50 μL) comprised 5 μL of denatured RNA, 1 μL of forward and reverse primers (10 pmol), and 43 μL of distilled water. The reaction profile was complementary DNA synthesis at 50°C for 30 min, followed by 35 cycles of denaturation-annealing-extension (95°C for 45 sec, 50°C for 45 sec, and 72°C for 45 sec), and a final extension at 72°C for 10 min. RT-PCR products were photo-documented in 2.0% agarose gels containing nucleic acid staining solution of RedSafe Nucleic Acid Staining Solution (iNtRON Biotechnology, Korea). The 2 purified RT-PCR products were ligated into the pGEM-T Easy Vector System (Promega, USA) and submitted to Macrogen (Korea) for sequencing. The sequencing results confirmed that the plasmid contained the GFP gene.
3
Liver Gene Expression Analysis
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Analyzing the gene expression level is to confirm the protein expression level. Total RNA was extracted from the liver tissue using an RNA extraction kit (Bioneer, Daejeon, Korea) according to the manufacturer’s instructions. The RNA concentration and purity were measured using the μDrop Plate (NanoDrop, Thermo Fisher Scientific, Delaware, DE, USA). cDNA was synthesized from 1 µg of total RNA using the AccuPower RocketScript Cycle RT PreMix (dT20) (Bioneer, Daejeon, Korea). The randomly selected gene primers were designed using gallus gallus genes with Primer 3 software (v.0.4.0, https://bioinfo.ut.ee/primer3-0.4.0/ ) and are shown in Supplemental Table S1 . The reverse transcription quantitative polymerase chain reaction (RT-qPCR) was conducted using the SsoFast EvaGreen Supermix (Bio-Rad, Hercules, CA, USA) on a CFX96 real-time PCR detection system (Bio-Rad, California, USA). The RT-qPCR thermal cycle conditions were as follows: 95 °C for 5 min, followed by 40 cycles of 95 °C for 5 and 60 °C for 30 s. The relative gene expression levels were calculated with the 2−ΔΔCt method [14 (link)] and normalized against the level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
4
Detection of Rabies Virus RNA in Diverse Tissues
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Total RNA was extracted from the homogenates of seven tissue samples (cerebrum, cerebellum, midbrain, spleen, liver, kidney, and lymph node) with an RNA extraction kit (Bioneer, Daejeon, Korea) according to the manufacturer's protocol. The extracted RNA was eluted in 50 µL of RNase- and DNase-free water. Reverse transcriptase-polymerase chain reaction (RT-PCR) was carried out to detect RABV genomic sequences using specific primer sets (RABVDNF and RABVDNR) that amplify the N gene of RABV (Table 1 ). RT-PCR was performed in a TPersonal 48 thermal cycler (Biometra, Horsham, PA, USA) with a reaction mixture containing 5 µL of denatured RNA, 1 µL of each primer (50 pmol), 5 µL of 5× buffer (12.5 mM MgCl2), 1 µL of dNTP mix, 1 µL of an enzyme mix (reverse transcriptase and Taq polymerase), and 11 µL of distilled water. The cycling program was as follows: cDNA synthesis at 42℃ for 30 minutes, followed by 45 cycles of 95℃ for 15 seconds, 55℃ for 15 seconds, 72℃ for 15 seconds, and a final extension at 72℃ for 5 minutes. The RT-PCR products were visualized by electrophoresis in 1.8% agarose gels containing ethidium bromide. Samples showing a 467 bp band were considered positive.
5
Nucleic Acid Extraction Protocols
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RNA was extracted from buffy coats of the blood samples, reference and archival strains, uninfected MDBK and RBK cell lines, and FMDV using the RNA extraction kit (Bioneer; South Korea) following the manufacturer’s instructions.
DNA was extracted from M. bovis, E. coli O157:H7, P. multocida, BLV-FLK, and BoHV-1 using the DNA extraction kit (Bioneer; South Korea) following the manufacturer’s instructions. All the extracted nucleic acids were stored at − 80 °C until used.
DNA was extracted from M. bovis, E. coli O157:H7, P. multocida, BLV-FLK, and BoHV-1 using the DNA extraction kit (Bioneer; South Korea) following the manufacturer’s instructions. All the extracted nucleic acids were stored at − 80 °C until used.
6
Total RNA Extraction and qRT-PCR Analysis
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Total RNA was isolated by an RNA extraction kit (Bioneer, Daejeon, Republic of Korea) in accordance with the procedures provided in the kit. An additional DNA digestion step was applied to ensure DNA was free from the extracted RNA using a Turbo DNA-free kit (Invitrogen, Carlsbad, CA, USA). The DNA-free RNA was confirmed by end-point PCR with 35 cycles, targeting the control gene gapdh. 2X OneStep qRT-PCR Master Mix (Biofact, Daejeon, Republic of Korea) was used to amplify the target genes using a Real-Time PCR System (Applied Biosystems, Waltham, Massachusetts, USA). Each primer set is listed in Table S3 . The specificity of the primer sets was examined by a melting curve. The expression levels of target genes were normalized to the internal gene gapdh (ETAC_06975), and the relative expression of target genes was calculated by Livak’s method.
7
Gene Expression Analysis by qRT-PCR
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Total RNA was extracted using RNA Extraction Kit (Bioneer, Daejeon, South Korea) according to the manufacturer’s instructions. The cDNA was synthesized using the CellScript™ cDNA Master Mix (CellSafe, Yongin, South Korea). The mixture with cDNA, SYBR Green (Toyobo, Osaka, Japan), forward primer, reverse primer was subjected to 35 cycles of PCR amplification using the following cycling conditions: denaturation at 95 °C for 5 s, annealing at 55 °C for 10 s, and extension at 72 °C for 30 s. The expression level of each mRNA was normalized to that of GAPDH. All primer sequences used in this study are listed in supplementary Table 1 .
8
Nucleic Acid Extraction for Viral and Bacterial Pathogens
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RNA was extracted from buffy coats of the blood samples, reference and archival strains, uninfected MDBK and RBK cell lines, and FMDV using the RNA extraction kit (Bioneer; South Korea) following the manufacturer's instructions.
DNA was extracted from M. bovis, E. coli O157:H7, P. multocida, BLV-FLK, and BoHV-1 using the DNA extraction kit (Bioneer; South Korea) following the manufacturer's instructions. All the extracted nucleic acids were stored at -80 °C until used.
DNA was extracted from M. bovis, E. coli O157:H7, P. multocida, BLV-FLK, and BoHV-1 using the DNA extraction kit (Bioneer; South Korea) following the manufacturer's instructions. All the extracted nucleic acids were stored at -80 °C until used.
9
Liver RNA Extraction and qPCR Analysis
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Total RNA was extracted from the liver tissue using an RNA extraction kit (Bioneer, Daejeon, Korea) according to the manufacturer's instructions. The RNA concentration and purity were measured using the µDrop Plate (NanoDrop, Thermo Fisher Scienti c). cDNA was synthesized from 1 µg of total RNA using the AccuPower RocketScript Cycle RT PreMix (dT20) (Bioneer). The randomly selected gene primers were designed with Primer 3 software (v.0.4.0) and are shown in Supplemental Table S1. The reverse transcription quantitative polymerase chain reaction (RT-qPCR) was conducted using the SsoFast EvaGreen Supermix (Bio-Rad, Hercules, CA, USA) on a CFX96 real-time PCR detection system (Bio-Rad). The RT-qPCR thermal cycle conditions were as follows: 95℃ for 5 min, followed by 40 cycles of 95℃ for 5 and 60℃ for 30 s. The relative gene expression levels were calculated with the 2 -ΔΔCt method {Livak, 2001 #131} and normalized against the level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
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