The cellular total RNA was isolated with mirVana™ miRNA isolation kit according to the manufacturer’s instruction. The reverse transcription of miRNAs was performed with the Applied Biosystems® TaqMan® MicroRNA Reverse Transcription kit and Applied Biosystems® 5x RT primer. The quantitative PCR amplifications of samples were done via Applied Biosystems® TaqMan® Universal PCR Master Mix, No AmpErase® UNG and 20x TaqMan small RNA Assay with protocol provided.
Rt primer
Manufactured by Thermo Fisher Scientific
The 5x RT primer is a reverse transcription primer used in the preparation of complementary DNA (cDNA) from RNA. It is a key component in the process of converting RNA into cDNA, which can then be utilized for various downstream applications, such as quantitative PCR analysis and gene expression studies.
Automatically generated - may contain errors
Lab products found in correlation
No amperase ung, by Thermo Fisher Scientific (2 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Taqman small rna assay, by Thermo Fisher Scientific (2 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Poly a polymerase, by Thermo Fisher Scientific (1 mentions)
Protoscript 2 reaction buffer, by New England Biolabs (1 mentions)
Lightcycler 480 2, by Roche (1 mentions)
Protoscript 2 reverse transcriptase, by New England Biolabs (1 mentions)
Mmlv reverse transcriptase, by Illumina (1 mentions)
Geneamp pcr system 9700, by Thermo Fisher Scientific (1 mentions)
Taqman probe, by Thermo Fisher Scientific (1 mentions)
6 protocols using rt primer
1
miRNA Isolation and Quantification
2
miRNA Isolation and Quantification
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The cellular total RNA was isolated with mirVana™ miRNA isolation kit accordingto the manufacturer's instruction. The reverse transcription of miRNAs was performed with the AppliedBiosystems® TaqMan® MicroRNA Reverse Transcription kit and Applied Biosystems® 5x RT primer. The quantitativePCR amplifications of samples were done via Applied Biosystems® TaqMan® Universal PCR Master Mix,No AmpErase® UNG and 20x TaqMan small RNA Assay with protocol provided.
3
Directed RNA Library Synthesis and Selection
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All DNA sequences (Supplementary Materials and Methods ) were obtained from IDT (Integrated DNA Technologies, IA) and gel purified. Double-stranded DNA libraries were generated by polymerase chain reaction (PCR) using 5′- and 3′-primers and the Pfu DNA polymerase, extracted with phenol/chloroform and precipitated with 1/10 volume NaAc 3 M pH 5.3 and 3 volumes ethanol 95%. RNA libraries were prepared by in vitro transcription using T7 RNA polymerase in Transcription buffer (40 mM Tris pH 7.6, 50 mM dithiothreitol, 1 mM spermidine, 0.1% triton, 7.5 mM MgCl2 and 4 mM of each adenosine triphosphate, cytidine triphosphate, uridine triphosphate and guanosine triphosphate) and incubation for 90 min at 37°C. To improve ribozyme cleavage of the active population, the reaction was incubated for an additional 1–20 min (20, 10, 10, 5, 2 and 1 min for cycles 1, 2, 3, 4, 5 and 6, respectively) after addition of 17.5 mM MgCl2. The large cleavage product was purified by denaturing gel electrophoresis [10% acrylamide:bisacrylamide (19:1) and 7 M urea]. The RNA product was then reverse transcribed using Superscript III reverse transcriptase with the RT-primer, according to the manufacturer’s protocol (Invitrogen, CA). The next rounds of selection were performed by following the same protocol, starting by regenerating the double-stranded DNA library, as described above.
4
Reverse Transcription and RT-qPCR Protocol
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Total (input) or AGO-immunoprecipitated RNA was reverse transcribed82 (link). Briefly, 100–500 ng of input or IP RNA was reverse transcribed in a final volume of 10 µL containing 2 µL 5× ProtoScript II reaction buffer (NEB), 25 µM ATP, 25 µM dNTPs, 50 µM RT primer (IK-44), 1 unit of poly(A) polymerase (Invitrogen) and 20 units of protoscript II reverse transcriptase (NEB). Reactions were incubated at 42 °C for 1 h followed by enzyme inactivation at 95 °C for 5 min. Real-time quantitative reverse transcriptase PCR (RT qPCR) was performed using a LightCycler 480 II (Roche) with SensiFAST SYBER No-ROX (Bioline Meridian Biosystems) using the gene-specific primers listed in Supplementary Table 2 . PCR was carried out in technical triplicates using the following cycling conditions: 95 °C for 3 min, followed by 45 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 10 s, and elongation at 72 °C for 20 s. A melting curve was generated at the end of the amplification in every run to confirm primer specificity. Threshold cycle (Ct) values were determined by calculating the second derivative maximum of three technical triplicates for each sample. Data were analysed using Prism-GraphPad Software v8.4.0.
5
Quantification of miRNA Expression
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Total RNA was extracted from frozen tissues using TRIzol reagent (Invitrogen). The RNA concentration was quantified by NanoDrop® ND-1000. Extracted RNA was reverse transcribed into complementary DNA using MMLV reverse transcriptase (Epicentre) and RT primers (Invitrogen). RT-PCR was carried out using TaqMan probes (Applied Biosystems) according to the manufacturer's instructions, with Gene Amp PCR System 9700 (Applied Biosystems). All reactions were run in triplicate, and average threshold cycle number (Ct) data of each miRNA was recorded. miRNAs expression in cells was normalized to U6 small noncoding RNA.
The ΔCt method and 2−ΔΔCt method were used for analysis. The ΔCt value was the difference between the Ct value of the specific miRNA and the Ct value of U6, ΔCt = Ct (miRNA) − Ct (U6). ΔΔCt = ΔCt (sample) − ΔCt (reference). 2−ΔCt represented miRNAs expression of each sample. 2−ΔΔCt represented the expression relative quotient (RQ) of target RNA to control RNA.
The ΔCt method and 2−ΔΔCt method were used for analysis. The ΔCt value was the difference between the Ct value of the specific miRNA and the Ct value of U6, ΔCt = Ct (miRNA) − Ct (U6). ΔΔCt = ΔCt (sample) − ΔCt (reference). 2−ΔCt represented miRNAs expression of each sample. 2−ΔΔCt represented the expression relative quotient (RQ) of target RNA to control RNA.
6
Quantitative Analysis of SLC1A5 mRNA
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Total RNA of HGC-27, MGC-803 cells of negative control group (empty plasmid) and SLC1A5-shRNA group cells were extracted by using TRIzol reagent (Invitrogen, U.S.A) according to the manufacture instructions. The first-strand cDNA was synthesized by using High-Capacity cDNA Reverse Transcription Kit (ABI, U.S.A). RT-primers of SLC1A5 mRNAs were designed and synthesized as follows: 5’-TGGTACGAAAATGTGGGCA-3’ (forward) and 5’-GTGCCCCAGCAGGCAGCACA-3’ (reverse) by Invitrogen Company. Real-time quantitative polymerase chain reaction (qRT-PCR) was performed according to TaqMan Gene Expression Assays protocol (ABI, U.S.A). The PCR program was set as follow: 95°C for 10 min, followed by 32 cycles of 95°C for 15 s, 60°C for 30 s, 72°C for 45 s.
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