For the evaluation of mRNA expression, RNA was extracted using RNA isolation kits according to the manufacturer's instructions (Tian Gen, China). Total RNA (~ 50 ng) was subjected to reverse transcription using the following stem-loop primers (RiboBio, Guangzhou, China) to generate the first-strand cDNA: CD133, F: 5′-AAGACCCATTTCCCTTGAGTTT-3′, R:5′-TTCATAGCCCCAGGAGTGTTAT-3′; ALDH, F:5′-AAAATGTCTCCATCACTTGG-3′, R:5′-AAGTCTTTGCCAATGCAT AC-3′; Oct-4, F: 5'-CTCACCCTGGGCGTTCTCT-3', R: 5'-AGGCCTCGAAGCG ACAGA-3'. Then the cDNA products were used for Eva Green-quantitative real-time PCR reaction with the above primers on iQTM 5 Real Time PCR Detection System (Bio-Rad, USA). PCR reactions were conducted at 95°C for 20 s followed by 40 cycles of 95°C for 10 s, 60°C for 20 s and 70°C for 7 s. Quantitative expression data were analyzed using the iCycler iQTM 5 software and reported as normalized fold expression relative to a β-actin as internal control (n = 3).
Rna isolation kit
Manufactured by Tiangen Biotech
Sourced in China
The RNA isolation kit is designed to efficiently extract and purify RNA from various biological samples. It utilizes a specialized protocol and reagents to isolate high-quality RNA, suitable for downstream applications such as RT-PCR, RNA sequencing, and gene expression analysis.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000, by Thermo Fisher Scientific (8 mentions)
Faststart universal sybr green master mix, by Roche (6 mentions)
Fastking one step rt pcr kit, by Tiangen Biotech (5 mentions)
Primescript rt reagent kit, by Takara Bio (4 mentions)
Realplex4, by Eppendorf (4 mentions)
Reverse transcription kit, by Takara Bio (4 mentions)
Superreal premix plus sybr green, by Tiangen Biotech (4 mentions)
Quantitative pcr detection system realplex 4, by Eppendorf (3 mentions)
M mlv reverse transcriptase, by Promega (3 mentions)
Gotaq qpcr master mix, by Promega (3 mentions)
Goscript reverse transcription system kit, by Promega (3 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Dnase 1, by Promega (2 mentions)
Reversely transcribed, by Tiangen Biotech (2 mentions)
Sybr green 1, by Tiangen Biotech (2 mentions)
Primescript 2, by Takara Bio (2 mentions)
Rna reverse transcription kit, by Beyotime (2 mentions)
Sybr green realtime pcr master mix, by Toyobo (2 mentions)
Lightcycle 96 system, by Roche (2 mentions)
Hiseq 2500, by Illumina (2 mentions)
87 protocols using rna isolation kit
1
Quantitative Analysis of mRNA Expression
2
Quantitative Expression Analysis of Cartilage Markers
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qRT-PCR was used to analyze the expression of type I, II and X collagen, aggrecan, Sox9, MMP-1, MMP-3 and TIMP-1. The sequences of the specific primers were designed according to specific genes and shown in Table 1 . Total intracellular RNA was extracted using RNA isolation kits (Tiangen Biotechnology; Beijing, China). Approximately 300 ng of total RNA was used as templates for the reverse transcription into cDNA using reverse transcription kit (Fermentas Company, Waltham, MA, USA). The qRT-PCR reactions were performed on a Quantitative PCR Detection System (Realplex 4, Eppendorf Corporation, Hauppauge, NY, USA) with a FastStart Universal SYBR Green Master (Mix, Roche Company, Grenzach, Germany) under the cycle conditions of 10 min at 95, 15 s at 95 °C and 1 min at 60 °C. The melting curve data were recorded to verify the PCR specificity. Each gene was analyzed in triplicate to diminish operation errors. The relative gene expression levels were calculated using the 2−ΔΔCt method according to glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
3
Comprehensive Analysis of Osteogenic Gene Expression
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Total RNA was subjected to isolation from hPDLSCs with the RNA Isolation kit (Tiangen Biotech Co., Ltd.). Determination of RNA quantification was undertaken with a NanoDrop 2000 spectrophotometer (NanoDrop Technologies). Reverse transcription of RNA to cDNA was done as instructed in the reverse transcription kit (Yeasen Biotech). A standard SYBR Green PCR kit (Takara Bio, Inc.) was used to amplify target fragments. The cDNA fragments of the GAPDH and U6 genes were employed for internal controls. The sequences of designed primer were clearly listed in Table 1 . Calculation of individual relative RNA levels was done utilizing the 2−ΔΔCq method [24 ].
The primer sequences for RT-qPCR used in the study
| Name | Sequences |
|---|---|
| OIP5-AS1 | Forward 5’-TGCGAAGATGGCGGAGTAAG-3’ |
| OCN | Forward 5’-CCCAGGCGCTACCTGTATCAA-3’ |
| OPN | Forward 5’-AGACCTGACATCCAGTACCCTG-3’ |
| RUNX2 | Forward 5’-TCCACACCATTAGGGACCATC-3’ |
| BMP2 | Forward 5’-AACACTGTGCGCAGCTTCC-3’ |
| GAPDH | Forward 5’-ACAACTTTGGTATCGTGGAAGG-3’ |
| miR-92a-3p | Forward 5’-CGCGTATTGCACTTGTCCC-3’ |
| U6 | Forward 5’-ATTGGAACGATACAGAGAAGATT-3’ |
4
NSCLC Patients' RNA Isolation and Quantification
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RNAs were isolated from blood samples of NSCLC patients using the commercial RNA isolation kit (TIANGEN Biotech, Beijing, China) and reversely transcribed to cDNAs (Applied Biosystems, Foster City, CA, USA). Amplification conditions were as follows: 95°C at 5 min and 35 cycles at 95°C for 10 s, 60°C for 30 s, and 72°C for 30 s. Primer sequences were as follows: AK021443, F: 5′-CTTGAACCCAGAAGACAGG-3′, R: 5′-ATGGAACATTAGAGGTAGCAC-3′; GAPDH, F: 5′-CGGATTTGGTCGTATTGGG-3′, R: 5′-GATTTTGGAGGGATCTCGC-3′.
5
Quantification of Gene Expression in E. coli
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Overnight cultures of WT and ΔrpoS were statically subcultured in DMEM at 28°C for 12 h, respectively. RNA samples were extracted with the RNA isolation kit (Tiangen) as previously described [13 (link)] and incubated with DNase I (Promega) for 30 min at 37°C to remove genomic DNA. RNA concentrations were measured with NanoDrop and 1 μg of each sample was used for reverse transcription with PrimeScript II 1st Strand cDNA Synthesis Kit (TaKaRa). The qRT-PCR was conducted on the Applied Biosystems 7500 real-time system (Applied Biosystems, Foster City, CA) in triplicate. The comparative CT (2-ΔΔCT) method was used to quantify the relative qualities of each transcript, and the housekeeping gyrB gene was used as an internal control. All the primers used are listed in S7 Table .
6
Transcriptional Analysis of Immune Responses
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J774A.1 cells were infected as described above, and the extracellular bacteria were harvested at 8 h post infection. E. piscicida were also cultured in medium and harvested as DMEM-cultured samples. RNA of both samples was extracted using an RNA isolation kit (Tiangen, Beijing, China). One microgram of each RNA sample was used for cDNA synthesis with the MMLV reverse transcriptase (ToYoBo, Tsuruga, Japan). RT-PCR was carried out on a FTC-200 detector (Funglyn Biotech, Shanghai, China) using the SYBR green real-time PCR kit (ToYoBo). Total RNA from whole fish was prepared as described above. The expression of TNF-α, IL-12, IL-10 and IFN-γ was determined using real-time PCR (ABI Step One qPCR). Each primer pair (Table S2) was designed using NCBI/Primer-BLAST. The expression of each gene was normalized to that of the β-actin transcript and expressed as -fold change relative to the expression seen in PBS-injected zebrafish. All quantitative PCRs were performed for three biological replicates, and the data for each sample were expressed relative to the expression level of the β-actin gene by using the 2 -ΔΔCT method.
7
RNA Isolation and RT-PCR Analysis
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RNA from AMs or lungs was isolated using RNA isolation kit (Tiangen) and reversely transcribed (Tiangen). The primers used for RT-PCR were listed in S1 Table . Actin was used as the internal control and the relative gene expression was calculated by the ΔΔCt quantification method.
8
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was extracted using RNA isolation kit (Tiangen Biotech) and subjected to cDNA synthesis using PrimeScript RT reagent Kit (Takara Bio). The cDNA was then used for the evaluation of the relative mRNA levels of the indentified gene, running in an ABI 7300 analyzer (Applied Biosystems). SYBR Green I (Tiangen Biotech) was used as the fluorescent probe. Primer sequences for RT-PCR are listed in the supplementary table 3 . The relative expression levels of the target genes were referred to as a housekeeping gene, GAPDH.
9
Comparative RT-qPCR Analysis of Gene Expression
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Samples of 5 donors, including Vp2S01::cat, Vp2S01ΔtrbE, Vp2S01ΔtraG, Vp2S01ΔtrbE::pRK415-trbE, and Vp2S01ΔtraG::pRK415-traG were collected. The total RNA per sample was treated with the RNA isolation kit (Tiangen) treated with DNase I following the manufacturer’s instructions. First-strand cDNA was synthesized from 500 ng total RNA according to instructions given in SPARKscript II RT Plus Kit (SparkJade Biotech). The real-time quantitative (RT-qPCR) assay was performed in a total volume of 20 μL, containing 1 μL of cDNA, 0.4 μL of each primer (10 μM), 10 μL of 2 × SYBR green qPCR Mix, and 8.2 μL RNase-free water (SparkJade Biotech). The analysis was conducted with Bio-Rad CFX Opus 96 Real-Time PCR Instrument (Bio-Rad) under the following conditions: 94°C for 3 min; 40 cycles at 94°C for 10 s, and 60°C for 30 s. The gene-specific primers are listed in Table 3 , and the relative expression of target genes was normalized by the 2−ΔΔCt method with the housekeeping gene gyrB as an internal control.
10
Quantitative RT-PCR Analysis of FAM50A Expression
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Total RNA was extracted from cells according to the instructions of the RNA Isolation Kit (Tiangen, Beijing, China). Then, 2 μg of total RNA was reverse transcribed into cDNA by the PrimeScript RT reagent kit for qRT-PCR (TaKaRa, Shiga, Japan). The reaction system of qRT-PCR was performed according to the instructions of the TB Green Premix Ex Taq™ II kit (TaKaRa, Shiga, Japan). GAPDH acted as the internal reference. The primers used were as follows: FAM50A forward 5′-TTCCGGGAGCTGGGAGATAA-3′, reverse 5′-CTCACCACCCACTCCAAGTC-3′, GAPDH forward 5′-CGGAGTCAACGGATTTGGTCGT-3′, reverse 5′-TCTCAGCCTTGACGGTGCCA-3′. The relative expression of genes was calculated by the 2−∆∆CT method [43 (link)].
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