To validate the key identified lncRNAs, we selected nine blood samples from sepsis patients (Table 2 ) and nine blood samples from healthy control subjects for qRT-PCR molecular validation. We extracted total RNA from the blood using the TRIpure reagent (ELK Biotechnology, EP013). M-MLV Reverse Transcriptase reagent Kit (ELK Biotechnology, EQ002) was used to synthesize cDNA according to the manufacturer’s instructions. Then, qRT-PCR was performed using the QuFast SYBR Green PCR Master Mix Kit (ELK Biotechnology, EQ001) to quantify the expression levels of lncRNAs, on a real-time PCR system (StepOnePlus; Applied Biosystems). GAPDH was used as the internal reference gene. Finally, we analyzed the data by the comparative quantitative cycle (Cq) (2-ΔΔCq) method. The research was approved by the Ethics Committee of Hubei Provincial Hospital of Traditional Chinese Medicine. All patients provided written informed consent for research on their specimens. Supplementary Table S1 shows the primer sequences used for qRT-PCR.
Tripure reagent
Manufactured by Elk Biotechnology
TRIpure reagent is a ready-to-use reagent designed for the isolation of high-quality total RNA from a variety of biological samples, including cells, tissues, and organs. It is based on the single-step RNA isolation method developed by Chomczynski and Sacchi.
Automatically generated - may contain errors
Lab products found in correlation
Enturbo sybr green pcr supermix, by Elk Biotechnology (2 mentions)
Steponeplus, by Thermo Fisher Scientific (1 mentions)
Qufast sybr green pcr master mix kit, by Elk Biotechnology (1 mentions)
Entilink 1st strand cdna synthesis kit, by Elk Biotechnology (1 mentions)
Stepone real time pcr system, by Elk Biotechnology (1 mentions)
Enturbo sybr green pcr supermix kit, by Elk Biotechnology (1 mentions)
Stepone real time pcr, by Thermo Fisher Scientific (1 mentions)
Stepone rt pcr thermocycler, by Thermo Fisher Scientific (1 mentions)
M mlv reverse transcriptase, by Elk Biotechnology (1 mentions)
8 protocols using tripure reagent
1
Validating Sepsis-Associated lncRNAs Using qRT-PCR
2
miR-200b-3p and HMGB3 Expression Quantification
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TRIpure Reagent (ELK Biotechnology) was used to extract the RNA from cells, and the EntiLink 1st Strand cDNA Synthesis Kit (ELK Biotechnology) was used to reverse-transcribe RNA into cDNA. Next, qPCR was conducted on the StepOne Real-Time PCR System using the EnTurbo SYBR Green PCR SuperMix Kit (ELK Biotechnology). U6 served as an internal control for miR-200b-3p. β-actin served as an internal control for HMGB3. The 2−ΔΔCq method was used for the quantification of data. U6, forward 5′-CTCGCTTCGGCAGCACAT-3′, reverse 5′-AACGCTTCACGAATTTGCGT-3′; miR-200b-3p, forward 5′-GGCCCTAATACTGCCTGGTA-3′, reverse 5′-CTCAACTGGTGTCGTGGAGTC-3′; β-actin, forward 5′-GTCCACCGCAAATGCTTCTA-3′, reverse 5′-TGCTGTCACCTTCACCGTTC-3′; HMGB3, forward 5′-CTATGATCGGGAAATGAAGGATTAT-3′, reverse 5′-TTCTCATACTTCTCCTTCAGCTTTG-3′.
3
Extraction and Quantification of RNA Expression
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TRIpure reagent (EP013, ELK Biotechnology) was used to extract total RNA from NP tissues and cultured cells. The primers used for qRT-PCR were as follows: homo GRP78, forward 5′-CATCACGCCGTCCTATGTCG-3′, and reverse 5′-CGTCAAAGACCGTGTTCTCG-3′. Homo CHOP, forward 5′-CCCTCACTCTCCAGATTCCAGTC-3′, and reverse 5′-CTAGCTGTGCCACTTTCCTTTCA-3′. Homo GAPDH, forward 5′-TCAAGAAGGTGGTGAAGCAGG-3′, and reverse 5′-TCAAAGGTGGAGGAGTGGGT-3′. GAPDH was used for normalization.
4
Quantitative RNA Expression Analysis
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According to the manufacturer’s guidelines, total RNA was extracted from each group of cells by TRI pure reagent (EP013, ELK Biotechnology, Wuhan, CHINA). The primers of genes were designed and synthesized by PINUOFEI Biotechnology Co., Ltd (Wuhan, CHINA). Then, EnTurbo™ SYBR Green PCR Super Mix (ELK Biotechnology, Wuhan, CHINA) and Step One™ Real-Time PCR (Life Technologies, USA) were used to determine the mRNA expression of genes. The following thermal circulator conditions were used: pre-denaturation at 95°C for 3 min, denaturation at 5°C for 10 s, and denaturation at 58°C for 30 s, and denaturation at 72°C for 30 s (denaturation for 40 cycles). The melting curve condition was the default setting of the instrument. GAPDH was used as internal reference to normalize the expression of gene. Relative quantification was determined by 2-ΔΔCT method.
5
RNA Extraction and qPCR Analysis
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RNA was extracted from harvested spinal cord tissues and PC12 cells using TRIpure reagent (EP013; ELK Biotechnology) according to the manufacturer's instructions. qPCR was performed with the use of QuFast SYBR Green PCR system (Life Technologies). Relative mRNA expression was calculated by the comparative ΔΔCT method using GAPDH as the housekeeping gene. The primers used in this study are shown in Table S1 .
6
Nfe2l2 Expression Analysis by qRT-PCR
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Total RNA was extracted from cells using TRIpure reagent (EP013, ELK Biotechnology) according to the manufacturer’s instructions. The following primers were used: rat Nfe2l2 (Gene ID: 83619) (F: 5’-TCCTCTGCTGCCATTAGTCA-3’, R: 5’-GTGCCTTCAGTGTGCTTCTG-3’) and rat Actb (Gene ID: 81,822) (F: 5’- TTCGTTGCCGGTCCACACCC-3’, R: 5’- GCTTTGCACATGCCGGAGCC-3’). β-Actin was used for normalization.
7
Liver mRNA Expression of Inflammatory Genes
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Total mRNA was extracted from liver tissues using a TRIpure reagent (ELK Biotechnology) according to the manufacturer's protocols. The synthesis of first-strand cDNA was performed using M-MLV Reverse Transcriptase (ELK Biotechnology). The expression levels of the target genes were measured with EnTurbo™ SYBR Green PCR SuperMix (ELK Biotechnology) using a StepOne™ RT-PCR thermocycler (Life Technologies). The mRNA level of each gene was normalized to the β-actin mRNA level of the same sample using the delta-delta CT method. All RT-PCR primer sequences are listed as follows: IL-1β 5′-ATGAAAGACGGCACACCCAC-3′ and 5′-GGTGCTGATGTACCAGTTGGG-3′; IL-6 5′-GCCAGAGTCATTCAGAGCAAT-3′ and 5′-CTTGGTCCTTAGCCACTCCT-3′; TNF-α 5′-CACCACGCTCTTCTGTCTACTG-3′ and 5′-GCTACGGGCTTGTCACTCG-3′; and β-actin 5′-CGTTGACATCCGTAAAGACCTC-3′ and 5′-TAGGAGCCAGGGCAGTAATCT-3′.
8
Quantifying Netrin-1 Expression in Spinal Cord, MSCs, Exosomes, and PC12 Cells
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TRIpure reagent (ELK Biotechnology, EP013) was used to extract total RNA from spinal cord tissues, MSCs, exosomes and PC12 cells. The following primers were used: netrin-1, forward primer, 5′-GTCTGAGAACTACCTGCAGTTCC-3′ and reverse primer, 5′-TACATTTTGCGGCACTGAGTG-3′; GAPDH, forward primer, 5′-AACAGCAACTCCCATTCTTCC-3′ and reverse primer, 5′- TGGTCCAGGGTTTCTTACTCC-3′. GAPDH was used for normalization.
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