The dorsal spinal cord tissues were removed 4 weeks after vehicle or MIA injection. The tissues were excised from the animals immediately following decapitation under 10% chloralic hydras anesthesia. Total RNA was isolated from the spinal cord specimens by using RNAeasyTM Animal RNA Isolation Kit with Spin Column (Beyotime Biotechnology, Nanjing, China). Aliquots of 2 μg total RNA were reverse transcribed into cDNA using ReverTra Ace-a-TM (Toyobo, Japan). The PCR mixture (10 μL) consisted of 1 μL diluted cDNA, 5 μL SYBR quantitative polymerase chain reaction (qPCR) mix (Takara, Japan), 1 μL of each primer, and 3 μL water. qPCR was performed using CFX96 system (Bio-Rad, UK), and the relative expression levels were quantified with CFX Manager software. The expression level of each gene was determined by the threshold cycle (CT). The amount of target mRNAs, normalized to the endogenous control (GAPDH), was obtained by 2−△△CT. Results of independent experiments were expressed as the percentage change over mRNA level of the control group. Specific sequence primers are listed in the follower: Mouse-5-HT2A forward 5′-AACCCCATTCACCATAGCCG-3′; reverse 5′-GGATATGGTCCACACCGCAA-3′. Mouse-GAPDH forward 5′-AGGTCGGTGTGAACGGATTTG-3′; reverse 5′-TGTAGACCATGTAGTTGAGGTCA-3′.
Rnaeasy animal rna isolation kit with spin column
Manufactured by Beyotime
Sourced in China
The RNAeasy™ Animal RNA Isolation Kit with Spin Column is a tool designed for the efficient extraction and purification of total RNA from various animal tissue samples. It utilizes a spin column-based method to isolate high-quality RNA that can be used in downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Lightcycler 480 system, by Roche (3 mentions)
Cfx96 system, by Bio-Rad (2 mentions)
Sybr quantitative polymerase chain reaction mix, by Takara Bio (2 mentions)
Revertra ace α, by Toyobo (2 mentions)
Primescript rt master mix, by Takara Bio (2 mentions)
Sybr green master mix, by Roche (2 mentions)
Beyofast sybr green one step qrt pcr kit, by Beyotime (2 mentions)
Transcriptor first strand cdna synthesis kit, by Roche (2 mentions)
Primescript rt reagent kit, by Takara Bio (2 mentions)
Prism 9, by GraphPad (1 mentions)
Tb green premix ex taq 2 tli rnaseh plus, by Takara Bio (1 mentions)
Universal sybr qpcr master mix, by Biosharp (1 mentions)
Reverse transcription kit with dsdnase, by Biosharp (1 mentions)
Sybr green pcr mastermix, by Solarbio (1 mentions)
Lightcycler 480 real time pcr system, by Roche (1 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (1 mentions)
Trizol, by Beyotime (1 mentions)
Beyort 2 first strand cdna synthesis kit, by Beyotime (1 mentions)
Beyofast sybr green qpcr mix, by Beyotime (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
35 protocols using rnaeasy animal rna isolation kit with spin column
1
Quantification of 5-HT2A Receptor mRNA
2
RNA Isolation and RT-qPCR for Breast Cancer
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Total RNA from breast cancer cells was isolated by using RNAeasy TM Animal RNA Isolation Kit with Spin Column (Beyotime Biotechnology, Shanghai, China). Total RNA from the serum of patients was isolated via Blood RNA Kit (Yeasen Biotechnology, Shanghai, China). The primer sequences were shown in Table S1 of the Supplementary materials. RNA extraction and RT-qPCR were performed using the HiScript II One Step RT-qPCR SYBR Green Kit-V22.1 (Vazyme Biotech, Nanjing, China). The relative expression was standardized as the internal control, and the 2 -∆∆CT method was used for calculation.
3
Spinal Cord 5-HT7 Receptor Expression
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The dorsal spinal cord tissues were removed 4 weeks after vehicle or MIA injection. The tissues were excised from mice immediately after the animals were anesthetized with 10% chloralic hydras and decapitated. Total RNA was isolated from the spinal cord tissues by using RNAeasyTM Animal RNA Isolation Kit with Spin Column (Beyotime Biotechnology, Nanjing, China). Aliquots of 2 μg total RNA were reverse transcribed into cDNA using ReverTraAce-a-TM (Toyobo, Japan). The PCR mixture (10 μl) consisted of 1 μl diluted cDNA, 5 μl SYBR quantitative polymerase chain reaction (qPCR) mix (Takara, Japan), 1 μl of each primer, and 3 μl of water. CFX96 system (Bio-Rad, United Kingdom) was used to perform qPCR, and the relative expression levels were quantified with CFX Manager software. The expression level of each gene was determined by the threshold cycle (CT). The amount of target mRNAs, normalized to the endogenous control (GAPDH), was obtained by 2–ΔΔCT. Results of independent experiments were expressed as the percentage change over mRNA level of the control group. Specific sequence primers are listed as follows: mouse-5-HT7 forward: 5′-GGCCTGAGAGAAGCGAGTTT-3′; reverse: 5′-TCACTCCGTTACCCCAAGGT-3′. Mouse-GAPDH forward: 5′-AGGTCGGTGTGAACGGATTTG-3′; reverse: 5′-TGTAGACCATGTAGTTGAGGTCA-3′.
4
Quantitative Real-Time PCR Analysis
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Fresh tissue samples were homogenized, and total RNA of tissues and monolayers were acquired using the RNAeasyTM Animal RNA Isolation Kit with Spin Column (R0024; Beyotime, Shanghai, China) according to the manufacturer’s guidelines. Transcript-specific primers were generated from GenBank, and primer specificity was verified using NCBI Primer Blast. Detailed primer sequences are shown in Table 1 . For Real-Time qPCR, a total of 2 μg RNA was used for reverse transcription using PrimeScriptTM RT master mix (RR036A; Takara, Shiga, Japan). Real-Time qPCR amplification was performed using the SYBR Green Master Mix (Roche; Bruxelles; Belgium) and the LightCycler480 system (Roche). Mean fold changes were calculated by averaging the three duplicate measurements and normalized to Gapdh. The 2–ΔΔCT method was used for calculation.
5
Quantitative Real-Time PCR Analysis
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Total RNA was extracted using the RNAeasy™ Animal RNA Isolation Kit with Spin Column (Beyotime, China) according to manufacturer’s instructions. Total RNA was reversely transcribed amplified using the BeyoFast™ SYBR Green One-Step qRT-PCR Kit (Beyotime, China). All real-time PCR primers were listed as following: AURKA, 5′-CTAACGGCTGAGCTCTTGGA-3′ and 5′-GAACCGACAGGGGACTTGAC-3′. CCNB1, 5′-ACCTTTGCACTTCCTTCGGA-3′ and 5′-TGTTCTTGACAGTCCATTCACCA-3′. CDK1, 5′-GCCCTTTAGCGCGGATCTAC-3′ and 5′-AGGAACCCCTTCCTCTTCACT-3′. TOP2A, 5′-CCGTCACCATGGAAGTGTCA-3′ and 5′-TGTCTGGGCGGAGCAAAATA-3′. CYP2B6, 5′-CCTCAACCTCAACACGCTCT-3′ and 5′-TTTGGCTCGGTCATGAAGCT-3′. CYP2C9, 5′-ACCAGCTGTGCTTCATTCCT-3′ and 5′-GCACAGTGAAACATAGGAAACTCTC-3′. CYP3A4, 5′-GCTTTCCTGCACATTAAGGAGAA AT-3′ and 5′-ATGGGCAAAGTCACAGTGGAT-3′. GAPDH, 5′-AGCCTCAAGATCATCAGC-3′ and 5′-GAGTCCTTCCACGATACC-3′. Relative mRNA expression levels of target genes were normalized by comparing to GAPDH, and calculated using the 2 − ΔΔCt method [41 (link)]. Expression data of selected genes were processed by GraphPad Prism 9, and analyzed by Student’s t-test. P-value less than 0.05 was regarded as the cutoff value of statistical significance.
6
RNA Extraction and qPCR Analysis of Colon Tissue
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Total RNA was extracted from colon tissues using the RNAeasy™ Animal RNA Isolation Kit with Spin Column (Beyotime, Shanghai, China), following the protocol of the kit. Briefly, the homogenized colon tissue was lysed and then centrifuged to take the supernatant, and finally total RNA was collected by the centrifugal column method. Then, we used a cDNA reverse transcription kit to reverse-transcribe the extracted RNA into cDNA. After that, TB Green™ Premix Ex Taq™ II (TliRNaseH Plus) (TaKaRa, Beijing, China) was added into the cDNA and quantified using the CFX Connect™ Fluorescent quantitative PCR detection system (BIO-RAD, Hercules, CA, USA). The related gene expression levels were objectively calculated using 2−ΔΔCt method, while beta-actin was used as the internal reference gene. Table S2 provides the primer sequence list of ZO-1, Claudin-1, Occludin, MAPK p38, JNK, NF-κB p65 and IκB-α.
7
Comprehensive RNA Extraction and Quantification
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Total RNA was extracted using the RNAeasy animal RNA isolation kit with spin column (R0036; Beyotime Biotechnology). RNA was then reverse transcribed into cDNA with the BeyoRT III first-strand cDNA synthesis kit with gDNA EZeraser (D7180M; Beyotime Biotechnology), as per the manufacturer’s instructions. Finally, DNA was amplified at least three times using the QuantStudio 6 flex real-time PCR system (Cata: 4485697; ThermoFisher Scientific), following the manufacturer’s recommendations.
8
Quantitative RNA Expression Analysis
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Total RNA was extracted using RNAeasy Animal RNA Isolation Kit with Spin Column (R0027, Beyotime, China). cDNA was synthesized from 1 μg of total RNA with Reverse Transcription Kit (with dsDNase) (BL699A, Biosharp, China). qRT-PCR was carried out with Universal SYBR qPCR Master Mix (BL697A, Biosharp, China) in a PikoReal Real-Time PCR machine. The relative expression level was determined by the 2-ΔCq method and normalized to Rpl19 expression. The primers used are listed in Table S3 .
9
Quantitative Real-Time PCR Analysis
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Four GC cells and GES-1 cells were harvested and total RNA was extracted with RNAeasy™ Animal RNA Isolation Kit with Spin Column (Beyotime Biotechnology, Shanghai, China). The Transcriptor First Strand cDNA Synthesis Kit (Roche, Basel, Switzerland) was used for reverse transcription. cDNA was mixed with 2×SYBR Green PCR Mastermix (Solarbio, Beijing, China) and then amplified and quantified by LightCycler 480 real-time PCR system (Roche, Basel, Switzerland). The level of mRNA expression was normalized to ACTB and 2−ΔΔCT method was used to evaluate relative gene expression.
10
Quantification of Apelin mRNA Expression
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Total RNA was extracted using RNAeasy™ Animal RNA Isolation Kit with Spin Column (Beyotime Biotechnology, Shanghai, China). The following primers were used to study the expressions of mouse GAPDH and apelin: mouse GAPDH forward: 5′-CCACTGTGGGCCACTTATACC-3′; mouse GAPDH reverse: 5′-CAGCCTTAGCCGAGCATTG-3′; mouse apelin forward: 5′-GGAATTCGGGACCATGAATCTGAGGCTCTG-3′; and mouse apelin reverse: 5′-ACTTGGCGAGCCCTTCAATC-3′. The qRT-PCR analysis was conducted using BeyoFast™ SYBR Green One-Step qRT-PCR Kit (Beyotime Biotechnology, Shanghai, China) on the platform of a CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The relative abundance of apelin was normalized by the level of GAPDH mRNA expression.
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