For qRT-PCR, total RNA (10 ng) was reverse transcribed into cDNA by using an All-in-One™ miRNA qRT-PCR Reagent Kit (GeneCopoeia) containing Poly A polymerase, a unique Oligo-dT Adaptor primer, and M-MLV Reverse Transcriptase. The quantitative PCR reactions were then carried out with an All-in-One™ qPCR Mix containing SYBR® Green, first-strand cDNA, specific miRNA primers, and the Universal Adaptor PCR Primer (GeneCopoeia) on a PRISM 7900HT system (Applied Biosystems). Every sample was analyzed in triplicate wells, and reactions excluding cDNA were used as negative controls. The thermal cycling conditions were: 95 °C for 10 min for a hot start, and then 40 cycles at 95 °C for 15 s, 60 °C for 20 s, and 72 °C for 10 s. U48 RNA was employed as the endogenous control. The qRT-PCR data were firstly normalized by U48 expression and then by a mean expression value of a given miRNA in the HSs. Therefore, the relative quantification of miRNA expression was presented as 2−ΔΔCt.
Universal adaptor pcr primer
Manufactured by GeneCopoeia
Sourced in United States
The Universal Adaptor PCR Primer is a versatile primer designed for use in polymerase chain reaction (PCR) amplification. It serves as a universal adaptor that can be used with a variety of DNA templates, allowing for the amplification of target sequences without the need for specific primer design.
Automatically generated - may contain errors
Lab products found in correlation
All in one mirna first strand cdna synthesis kit, by GeneCopoeia (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
All in one mirna qrt pcr detection kit, by GeneCopoeia (2 mentions)
Hiscript 2 q rt supermix, by Vazyme (2 mentions)
Cfx connect real time system, by Bio-Rad (2 mentions)
Chamq sybr color qpcr master mix, by Vazyme (2 mentions)
Prism 7900ht system, by Thermo Fisher Scientific (1 mentions)
All in one mirna qrt pcr reagent kit, by GeneCopoeia (1 mentions)
All in one qpcr mix, by GeneCopoeia (1 mentions)
All in onetm mirna qrt pcr detection kit, by GeneCopoeia (1 mentions)
Abi 7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Rnaprep pure ffpe kit, by Tiangen Biotech (1 mentions)
Naio4, by Merck Group (1 mentions)
Mirna first strand cdna synthesis kit, by GeneCopoeia (1 mentions)
Borate borax buffer, by Merck Group (1 mentions)
Evagreen, by Thermo Fisher Scientific (1 mentions)
All in onetm mirna first strand cdna synthesis kit, by GeneCopoeia (1 mentions)
8 protocols using universal adaptor pcr primer
1
Quantitative miRNA expression analysis
2
Quantification of miRNA Levels in DLBCL
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Total RNA was extracted with the Trizol reagent (Invitrogen). Total RNA from Paraffin-embedded tissues was isolated using RNAprep pure FFPE Kit (Cat # DP439, TianGen, China) according to the manufacturer’s instruction. DLBCL was confirmed and selected by pathologists. qRT-PCR was performed by investigators not knowing the histopathological and immunophenotypic characteristics of the human lymphoma specimen. All-in-OneTM miRNA qRT-PCR Detection kit (GeneCopoeia, Rockville, MD, USA) was used to quantify miRNA levels according to the manufacturer’s instructions. The small RNA U6 was used as internal control for miRNA detection. qRT-PCR was performed on an ABI 7500 real-time PCR system (Applied Biosystems). Relative miRNA level was calculated using the δCt method. All reactions were conducted in triplicate. Forward primers for miR-451, miR-144, and U6 were 5′-AAA CCG TTA CCA TTA CTG AGT T-3′, 5′-TAC AGT ATA GAT GAT GTA CT-3′ and 5′-CGC TTC GGC AGC ACA TAT AC-3′, respectively, and the Universal Adaptor PCR Primer (GeneCopoeia) was used as the reverse primer.
3
Irradiation-Induced miRNA Profiling in CRC
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The SW837 cells were plated in 100-mm dishes containing medium and 10% FBS for 24 h before IR and exposed to X-ray unit at a dose rate of 1.55 Gy/min. WT cells for control were cultured with previously irradiated medium and 10% FBS. Meantime, SW837 cells were exposed to IR (2Gy/day) for 7 days and the surviving cells were continue to culture until there was enough cells for analysis. miRNA qPCR arrays for profiling the expressions of CRC-related miRNAs and specific primers were designed by GeneCopoeia and added prior to 96 well plate (GeneCopoeia, Rockville, USA). Poly A Polymerase, RTase Mix, qPCR Mix, ROX Reference Dye, Universal Adaptor PCR Primer were purchased from GeneCopoeia (GeneCopoeia, Rockville, USA). RNU48 and U6 were detected and used as internal standards.
4
Small RNA Sequencing and qRT-PCR
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Small RNAs were suspended in 1× borate/borax buffer with or without NaIO4 (final concentration: 25 mM) (Sigma-Aldrich) and incubated for 30 min at room temperature in the dark. After adding 1/10 volume of 100% glycerol, samples were further incubated for 10 min at room temperature in the dark. After purification, RNA samples were precipitated using ethanol and used as input for Solexa sequencing by Illumina 3000 or qRT-PCR by all in one miRNA First Strand cDNA Synthesis Kit (GeneCopoeia, Guangzhou, China), containing poly(A) polymerase and RTase Mix, according to the manufacturer's instruction (20 (link)). After obtaining cDNAs, rTaq (Takara), dNTP (Takara), EvaGreen (Invitrogen), sense primer and Universal Adaptor PCR Primer (GeneCopoeia) (Supplemental Table S1) were added for qRT-PCR detection. The program was as follows: 95°C for 5 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Each cDNA sample was tested in triplicate.
5
Quantification of miRNA and mRNA Expression
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Total RNA was extracted as previously defined. For mature miRNAs, All-in-One™ miRNA First-Strand cDNA Synthesis Kit (Genecopoeia) was used to generate 10 μL cDNA. Then using miR-specific primers and universal adaptor PCR primers (Genecopoeia), qRT-PCR was performed with All-in-One™ miRNA qRT-PCR Detection Kit (Genecopoeia) on CFX Connect Real-Time System (Bio-Rad). For mRNA, the reverse transcription was carried out using HiScript II Q RT SuperMix (Vazyme), and qRT-PCR was performed with ChamQ™ SYBR Color qPCR Master Mix (Vazyme) on CFX Connect Real-Time System (Bio-Rad) [21 (link)]. Specific primers for qRT-PCR of mRNA were listed in Supplementary Data . The U6 small nuclear RNA or GAPDH was used as an endogenous control. Each experiment was repeated at least three times and the results were analyzed using 2-△△CT method.
6
Quantitative RT-PCR Analysis of miRNA
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Total RNA was isolated as described above. Reverse transcription of 10 μl purified cell culture medium RNA into cDNA was carried out using an All-in-OneTM miRNA First-Strand cDNA Synthesis Kit (Genecopoeia). Subsequent real-time PCR, using miR-specific primers and universal adaptor PCR primers (Genecopoeia), was performed with a Stratagene Mx3000p QPCR System (Genetimes). The reactions were incubated in a 96 well plate at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. All reactions were run in triplicate.
7
Quantitative miRNA Expression Analysis
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Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. All-in-One™ miRNA First-Strand cDNA Synthesis Kit (Genecopoeia) was used to transcribe 10 μL of purified RNA. Real-time PCR, using miR-specific primers and universal adaptor PCR primers (Genecopoeia), was performed with a Stratagene Mx3000P QPCR System (Genetimes Technology, Shanghai, China). The reactions were incubated in a 96 well plate at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s, and 60 °C for 1 min. All reactions were run in triplicate.
8
Quantification of miRNA and mRNA Levels
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Total RNA was extracted as previously de ned. For mature miRNAs, All-in-One™ miRNA First-Strand cDNA Synthesis Kit (Genecopoeia) was used to generate 10 µL cDNA. Then using miR-speci c primers and universal adaptor PCR primers (Genecopoeia), qRT-PCR was performed with All-in-One™ miRNA qRT-PCR Detection Kit (Genecopoeia) on CFX Connect Real-Time System (Bio-Rad). For mRNA, the reverse transcription was carried out using HiScript II Q RT SuperMix (Vazyme), and qRT-PCR was performed with ChamQ™ SYBR Color qPCR Master Mix (Vazyme) on CFX Connect Real-Time System (Bio-Rad) (21) . Speci c primers for qRT-PCR of mRNA were listed in Supplementary Data. The U6 small nuclear RNA or GAPDH was used as an endogenous control. Each experiment was repeated at least three times and the results were analyzed using 2 -△△CT method.
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